Unknown,Transcriptomics,Genomics,Proteomics

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A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation.

ABSTRACT: Analysis of the intergenic regions of Staphylococcus aureus by bioinformatics predicted multiple regulatory regions. From this analysis, we also characterized 11 novel noncoding RNAs (RsaA-K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma factor B (RsaA, D, and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other staphylococci, and RsaE is additionally present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE-mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C-rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism. Total RNA of the strain Staphylococcus aureus RN6390 (wild type) was co-hybridized in duplicate with the RsaX15 mutant strain (Staphylococcus aureus Lug1430). cDNA of RN6390 was labeled with Cy-5 ; cDNA of RN6390 was labeled with Cy-3. Hybridization, performed in Agilent proprietary buffer was performed for 17H at 60Â°C. Slides were then scanned in the Agilent scanner and extracted with Feature Extraction software. Background subtracted data were normalized for unequal incorporation or loading (LOWESS).

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Marie-JosÃ©e Cros

PROVIDER: E-GEOD-17135 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation.

Geissmann Thomas T Chevalier Clément C Cros Marie-Josée MJ Boisset Sandrine S Fechter Pierre P Noirot Céline C Schrenzel Jacques J François Patrice P Vandenesch François F Gaspin Christine C Romby Pascale P

Nucleic acids research 20091101 21

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA-K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) ...[more]

PMID: 19786493

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Project description:Understanding how pathogens respond to antimicrobial peptides, and how this compares to currently available antibiotics, is crucial to optimizing antibiotic therapy. Staphylococcus aureus has several known resistance mechanisms against human cationic antimicrobial peptides (CAMPs). We aim to determine how S. aureus responds to sheep and frog CAMPs, and whether this response is associated with resistance. Gene expression changes in Staphylococcus aureus Newman cells exposed to linear CAMPs were analyzed by DNA microarray. Three antimicrobial peptides were used in the analysis, two of them are derived from frog, temporin L and dermaseptin K4-S4(1-16), one is from sheep, ovispirin-1. The peptides induced the VraSR cell-wall regulon and several other genes which are also upregulated in cells treated with vancomycin and other cell wall-active antibiotics. In addition to this similarity, three genes/operons were particularly strongly induced by the peptides: vraDE, SA0205 and SAS016, encoding an ABC transporter, a putative membrane-bound lysostaphin-like peptidase and a small functionally unknown protein, respectively. Ovispirin-1 and dermaseptin K4-S4(1-16), which disrupt lipid bilayers by the carpet mechanism, were strong inducers of the vraDE operon. We show that high level induction by ovispirin-1 was dependent on the amide modification of the peptide C-terminus. This suggests that the amide group has a crucial role in the activation of the Aps sensory system, the regulator of vraDE. In contrast, temporin L, which disrupts lipid bilayers by forming pores, was clearly a weaker inducer of vraDE despite the C-terminal amide modification. Sensitivity testing with CAMPs and other antimicrobials suggested that VraDE is a transporter dedicated to resist bacitracin. We also showed that SA0205 belongs to the VraSR regulon. Furthermore, VraSR was shown to be important for resistance against a wide range of cell wall-active antibiotics and other antimicrobial agents including the amide-modified ovispirin-1, bacitracin, teicoplanin, cefotaxime and 10 other β-lactam antibiotics, chlorpromazine, thioridazine and EGTA. The effects of the three different antimicrobial peptides on gene expression of S. aureus Newman were studied by using whole genome oligo-DNA microarrays. Bacteria were grown in BHI medium to the early exponential phase (OD600=0.6) and antimicrobial peptides were added at sublethal concentrations. Samples were taken for RNA isolations after treating the cultures with the peptides for 10 minutes. Control cultures without peptide additions were treated similarly and in parallel.

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A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation.

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A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation.

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