The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus.

RsaE is a regulatory RNA highly conserved amongst Firmicutes that lowers the amount of mRNAs associated with the TCA cycle and folate metabolism. A search for new RsaE targets in Staphylococcus aureus revealed that in addition to previously described substrates, RsaE down-regulates several genes associated with arginine catabolism. In particular, RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. The faster growth rate of ΔrsaE compared to its parental strain in media containing amino acids as sole carbon source points to an underlying role for RsaE in amino acid catabolism. Collectively, the data support a model in which RsaE acts as a global regulator of functions associated with metabolic adaptation.

Assessment of Bona Fide sRNAs in Staphylococcus aureus.

Bacterial regulatory RNAs have been extensively studied for over a decade, and are progressively being integrated into the complex genetic regulatory network. Transcriptomic arrays, recent deep-sequencing data and bioinformatics suggest that bacterial genomes produce hundreds of regulatory RNAs. However, while some have been authenticated, the existence of the others varies according to strains and growth conditions, and their detection fluctuates with the methodologies used for data acquisition and interpretation. For example, several small RNA (sRNA) candidates are now known to be parts of UTR transcripts. Accurate annotation of regulatory RNAs is a complex task essential for molecular and functional studies. We defined bona fide sRNAs as those that (i) likely act in trans and (ii) are not expressed from the opposite strand of a coding gene. Using published data and our own RNA-seq data, we reviewed hundreds of Staphylococcus aureus putative regulatory RNAs using the DETR'PROK computational pipeline and visual inspection of expression data, addressing the question of which transcriptional signals correspond to sRNAs. We conclude that the model strain HG003, a NCTC8325 derivative commonly used for S. aureus genetic regulation studies, has only about 50 bona fide sRNAs, indicating that these RNAs are less numerous than commonly stated. Among them, about half are associated to the S. aureus sp. core genome and a quarter are possibly expressed in other Staphylococci. We hypothesize on their features and regulation using bioinformatic approaches.

Finding sRNA-associated phenotypes by competition assays: An example with Staphylococcus aureus.

Bacteria optimize their fitness in response to a changing environment by tight regulation of gene expression. Regulation can be controlled at both transcriptional and post-transcriptional levels via key players such as sigma factors, regulatory proteins and regulatory RNAs. The identification of phenotypes associated with gene deletions is the established method for finding gene functions but may require testing many conditions for each studied mutant. As regulatory RNAs often contribute to fine-tuning gene expression, phenotypes associated with their inactivation are often weak and difficult to detect. Nevertheless, minor phenotypes conferring modest advantages, may allow bacteria to emerge after some generations under selective pressure. A strategy employing DNA barcodes can be used to perform competition experiments between mutants and to monitor fitness associated with mutations in different growth conditions. We combined this strategy with deep sequencing to study regulatory RNAs in Staphylococcus aureus, a major opportunistic pathogen.

Mechanistic study of base-pairing small regulatory RNAs in bacteria.

In all three kingdoms of life, RNA is not only involved in the expression of genetic information, but also carries out extremely diverse cellular functions. This versatility is essentially due to the fact that RNA molecules can exploit the power of base pairing to allow them to fold into a wide variety of structures through which they can perform diverse roles, but also to selectively target and bind to other nucleic acids. This is true in particular for bacterial small regulatory RNAs that act by imperfect base-pairing with target mRNAs, and thereby control their expression through different mechanisms. Here we outline an overview of in vivo and in vitro approaches that are currently used to gain mechanistic insights into how these sRNAs control gene expression in bacteria.