Anne-Catherine Helfer

Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression.

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.

A nitric oxide regulated small RNA controls expression of genes involved in redox homeostasis in Bacillus subtilis.

RsaE is the only known trans-acting small regulatory RNA (sRNA) besides the ubiquitous 6S RNA that is conserved between the human pathogen Staphylococcus aureus and the soil-dwelling Firmicute Bacillus subtilis. Although a number of RsaE targets are known in S. aureus, neither the environmental signals that lead to its expression nor its physiological role are known. Here we show that expression of the B. subtilis homolog of RsaE is regulated by the presence of nitric oxide (NO) in the cellular milieu. Control of expression by NO is dependent on the ResDE two-component system in B. subtilis and we determined that the same is true in S. aureus. Transcriptome and proteome analyses revealed that many genes with functions related to oxidative stress and oxidation-reduction reactions were up-regulated in a B. subtilis strain lacking this sRNA. We have thus renamed it RoxS. The prediction of RoxS-dependent mRNA targets also suggested a significant enrichment for mRNAs related to respiration and electron transfer. Among the potential direct mRNA targets, we have validated the ppnKB mRNA, encoding an NAD+/NADH kinase, both in vivo and in vitro. RoxS controls both translation initiation and the stability of this transcript, in the latter case via two independent pathways implicating RNase Y and RNase III. Furthermore, RNase Y intervenes at an additional level by processing the 5′ end of the RoxS sRNA removing about 20 nucleotides. Processing of RoxS allows it to interact more efficiently with a second target, the sucCD mRNA, encoding succinyl-CoA synthase, thus expanding the repertoire of targets recognized by this sRNA.

Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

Enzymatic probing and lead(II)-induced cleavages have been developed to study the secondary structure of RNA molecules either free or engaged in complex with different ligands. Using a combination of probes with different specificities (unpaired vs. paired regions), it is possible to get information on the accessibility of each nucleotide, on the binding site of a ligand (noncoding RNAs, protein, metabolites), and on RNA conformational changes that accompanied ligand binding or environmental conditions (temperature, pH, ions, etc.). The detection of the cleavages can be conducted by two different ways, which are chosen according to the length of the studied RNA. The first method uses end-labeled RNA molecules and the second one involves primer extension by reverse transcriptase. We provide here an experimental procedure that was designed to map the structure of mRNA and mRNA-sRNA interaction in vitro.

Two novel members of the LhrC family of small RNAs in Listeria monocytogenes with overlapping regulatory functions but distinctive expression profiles

ABSTRACT Multicopy small RNAs (sRNAs) have gained recognition as an important feature of bacterial gene regulation. In the human pathogen Listeria monocytogenes, 5 homologous sRNAs, called LhrC1-5, control gene expression by base pairing to target mRNAs though 3 conserved UCCC motifs common to all 5 LhrCs. We show here that the sRNAs Rli22 and Rli33-1 are structurally and functionally related to LhrC1-5, expanding the LhrC family to 7 members, which makes it the largest multicopy sRNA family reported so far. Rli22 and Rli33-1 both contain 2 UCCC motifs important for post-transcriptional repression of 3 LhrC target genes. One such target, oppA, encodes a virulence-associated oligo-peptide binding protein. Like LhrC1-5, Rli22 and Rli33-1 employ their UCCC motifs to recognize the Shine-Dalgarno region of oppA mRNA and prevent formation of the ribosomal complex, demonstrating that the 7 sRNAs act in a functionally redundant manner. However, differential expression profiles of the sRNAs under infection-relevant conditions suggest that they might also possess non-overlapping functions. Collectively, this makes the LhrC family a unique case for studying the purpose of sRNA multiplicity in the context of bacterial virulence.

A method to map changes in bacterial surface composition induced by regulatory RNAs in Escherichia coli and Staphylococcus aureus.

We have adapted a method to map cell surface proteins and to monitor the effect of specific regulatory RNAs on the surface composition of the bacteria. This method involves direct labeling of surface proteins of living bacteria using fluorescent dyes and a subsequent separation of the crude extract by 2D gel electrophoresis. The strategy yields a substantial enrichment in surface proteins over cytoplasmic proteins. We validated this method by monitoring the effect of the regulatory RNA MicA in Escherichia coli, which regulates the synthesis of several outer membrane proteins, and highlighted the role of Staphylococcus aureus RNAIII for the maintenance of cell wall integrity.

In vivo mapping of RNA-RNA interactions in Staphylococcus aureus using the endoribonuclease III.

Ribonucleases play key roles in gene regulation and in the expression of virulence factors in Staphylococcus aureus. Among these enzymes, the double-strand specific endoribonuclease III (RNase III) is a key mediator of mRNA processing and degradation. Recently, we have defined, direct target sites for RNase III processing on a genome-wide scale in S. aureus. Our approach is based on deep sequencing of cDNA libraries obtained from RNAs isolated by in vivo co-immunoprecipitation with wild-type RNase III and two cleavage-defective mutants. The use of such catalytically inactivated enzymes, which still retain their RNA binding capacity, allows the identification of novel RNA substrates of RNase III. In this report, we will summarize the diversity of RNase III functions, discuss the advantages and the limitations of the approach, and how this strategy identifies novel mRNA targets of small non-coding RNAs in S. aureus.

sRNA-mediated activation of gene expression by inhibition of 5'-3’ exonucleolytic mRNA degradation

Post-transcriptional control by small regulatory RNA (sRNA) is critical for rapid adaptive processes. sRNAs can directly modulate mRNA degradation in Proteobacteria without interfering with translation. However, Firmicutes have a fundamentally different set of ribonucleases for mRNA degradation and whether sRNAs can regulate the activity of these enzymes is an open question. We show that Bacillus subtilis RoxS, a major trans-acting sRNA shared with Staphylococus aureus, prevents degradation of the yflS mRNA, encoding a malate transporter. In the presence of malate, RoxS transiently escapes from repression by the NADH-sensitive transcription factor Rex and binds to the extreme 5’-end of yflS mRNA. This impairs the 5’-3’ exoribonuclease activity of RNase J1, increasing the half-life of the primary transcript and concomitantly enhancing ribosome binding to increase expression of the transporter. Globally, the different targets regulated by RoxS suggest that it helps readjust the cellular NAD+/NADH balance when perturbed by different stimuli. DOI: http://dx.doi.org/10.7554/eLife.23602.001

Various checkpoints prevent the synthesis of Staphylococcus aureus peptidoglycan hydrolase LytM in the stationary growth phase

ABSTRACT In Staphylococcus aureus, peptidoglycan metabolism plays a role in the host inflammatory response and pathogenesis. Transcription of the peptidoglycan hydrolases is activated by the essential 2-component system WalKR at low cell density. During stationary growth phase, WalKR is not active and transcription of the peptidoglycan hydrolase genes is repressed. In this work, we studied regulation of expression of the glycylglycine endopeptidase LytM. We show that, in addition to the transcriptional regulation mediated by WalKR, the synthesis of LytM is negatively controlled by a unique mechanism at the stationary growth phase. We have identified 2 different mRNAs encoding lytM, which vary in the length of their 5′ untranslated (5′UTR) regions. LytM is predominantly produced from the WalKR-regulated mRNA transcript carrying a short 5′UTR. The lytM mRNA is also transcribed as part of a polycistronic operon with the upstream SA0264 gene and is constitutively expressed. Although SA0264 protein can be synthesized from the longer operon transcript, lytM cannot be translated because its ribosome-binding site is sequestered into a translationally inactive secondary structure. In addition, the effector of the agr system, RNAIII, can inhibit translation of lytM present on the operon without altering the transcript level but does not have an effect on the translation of the upstream gene. We propose that this dual regulation of lytM expression, at the transcriptional and post-transcriptional levels, contributes to prevent cell wall damage during the stationary phase of growth.