Philippe Bouloc

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.

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

BackgroundThe 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.ResultsWe 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.ConclusionOur 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.

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.

Down-regulation of Porins by a Small RNA Bypasses the Essentiality of the Regulated Intramembrane Proteolysis Protease RseP in Escherichia coli

Adaptation to extracytoplasmic stress in Escherichia coli depends on the activation of σE, normally sequestered by the membrane protein RseA. σE is released in response to stress through the successive RseA cleavage by DegS and the RIP protease RseP. σE and proteases that free it from RseA are essential. We isolated a multicopy suppressor that alleviated RseP and DegS requirement. The suppressor encodes a novel small RNA, RseX. Its activity required the RNA-binding protein Hfq. We used the property that small RNAs are often involved in RNA-RNA interactions to capture RseX putative partners; ompA and ompC mRNA, which encode two major outer membrane proteins, were identified. RseX activity was shown to confer an Hfq-dependent coordinate OmpA and OmpC down-regulation. Because RseP is shown to be no longer essential in a strain lacking OmpA and OmpC, we conclude that RseP, which is required for normal σE activation, prevents toxicity due to the presence of two specific outer membrane proteins that are down-regulated by RseX.

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.

Single-pass classification of all noncoding sequences in a bacterial genome using phylogenetic profiles

Identification and characterization of functional elements in the noncoding regions of genomes is an elusive and time-consuming activity whose output does not keep up with the pace of genome sequencing. Hundreds of bacterial genomes lay unexploited in terms of noncoding sequence analysis, although they may conceal a wide diversity of novel RNA genes, riboswitches, or other regulatory elements. We describe a strategy that exploits the entirety of available bacterial genomes to classify all noncoding elements of a selected reference species in a single pass. This method clusters noncoding elements based on their profile of presence among species. Most noncoding RNAs (ncRNAs) display specific signatures that enable their grouping in distinct clusters, away from sequence conservation noise and other elements such as promoters. We submitted 24 ncRNA candidates from Staphylococcus aureus to experimental validation and confirmed the presence of seven novel small RNAs or riboswitches. Besides offering a powerful method for de novo ncRNA identification, the analysis of phylogenetic profiles opens a new path toward the identification of functional relationships between co-evolving coding and noncoding elements.

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA‐tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C‐terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.

Escherichia coli metabolism in space

Cultures of the bacterium Escherichia coli were grown in the orbiting Biocosmos 2044 satellite in order to evaluate the effects of the space environment--weightlessness and heavy particle radiation--on growth parameters and energy metabolism, which have previously been reported to be affected, and on induction of the SOS response, which reflects DNA damage to the cell. We found no differences between the flight samples and control ground cultures in the growth yield per gram of carbon, in mean cell mass (from which we deduce that the growth rate was unaltered) or in the level of expression of the SOS response. These observations indicate that free-growing bacterial cells do not expend significant energy fighting gravity and that cosmic radiation within a space capsule does not produce significant levels of DNA damage.

An assessment of bacterial small RNA target prediction programs

Most bacterial regulatory RNAs exert their function through base-pairing with target RNAs. Computational prediction of targets is a busy research field that offers biologists a variety of web sites and software. However, it is difficult for a non-expert to evaluate how reliable those programs are. Here, we provide a simple benchmark for bacterial sRNA target prediction based on trusted E. coli sRNA/target pairs. We use this benchmark to assess the most recent RNA target predictors as well as earlier programs for RNA-RNA hybrid prediction. Moreover, we consider how the definition of mRNA boundaries can impact overall predictions. Recent algorithms that exploit both conservation of targets and accessibility information offer improved accuracy over previous software. However, even with the best predictors, the number of true biological targets with low scores and non-targets with high scores remains puzzling.

Dispensable PDZ domain of Escherichia coli YaeL essential protease.

In Escherichia coli, adaptation to extra‐cytoplasmic stress relies on σE activation to induce a rescue pathway. Under non‐stressed conditions, σE is sequestered by the inner membrane protein RseA. Extra‐cytoplasmic stress activates DegS‐dependent cleavage of RseA, rendering RseA sensitive to further degradation by the YaeL protease. YaeL contains two motifs characteristic of a family of metallo‐proteases, as well as a periplasmic PDZ domain. We report results of mutational analyses of the YaeL domains. Surprisingly, expression in a strain depleted for wild‐type YaeL or YaeL variants having a 40 amino acid deletion of the PDZ domain or amino acid substitutions of conserved amino acids of the YaeL PDZ domain did not affect cell viability. The proteolytic activity against RseA of these YaeL variants became independent of DegS. These observations suggest that the YaeL PDZ domain exerts a negative control on YaeL activity. Rather than being involved in substrate recognition, the PDZ domain of YaeL is likely to act as an inhibitor of proteolytic activity.