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

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

Staphylococcus aureus pigmentation is not controlled by Hfq

International audienceOBJECTIVE: The golden color of Staphylococcus aureus is due to the synthesis of carotenoid pigments. In Gram-negative bacteria, Hfq is a global posttranscriptional regulator, but its function in S. aureus remains obscure. The absence of Hfq in S. aureus was reported to correlate with production of carotenoid pigment leading to the conclusion that Hfq was a negative regulator of the yellow color. However, we reported the construction of hfq mutants in several S. aureus strains and never noticed any color change; we therefore revisited the question of Hfq implication in S. aureus pigmentation. RESULTS: The absence or accumulation of Hfq does not affect S. aureus pigmentation

Staphylococcus aureus staphyloxanthin expression is not controlled by Hfq

ABSTRACT Objective The golden color of Staphylococcus aureus is due to the synthesis of carotenoid pigments. In Gram-negative bacteria, Hfq is a global posttranscriptional regulator, but its function in S. aureus remains obscure. The absence of Hfq in S. aureus was reported to correlate with production of carotenoid pigment leading to the conclusion that Hfq was a negative regulator of the yellow color. However, we reported the construction of hfq mutants in several S. aureus strains and never noticed any color change; we therefore revisited the question of Hfq implication in S. aureus pigmentation. Results The absence or accumulation of Hfq does not affect S. aureus pigmentation

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

Protein Aggregation in a Mutant Deficient in YajL, the Bacterial Homolog of the Parkinsonism-associated Protein DJ-1

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase β subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant

Dynamic insights on transcription initiation and RNA processing during bacterial adaptation

International audienceTranscription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously-described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity; i) constant and independent of RNA levels, ii) increased once RNA has accumulated, and iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes, and can serve as blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control