Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5'-Caps, but No CoAlation Marks.
Ontology highlight
ABSTRACT:
SUBMITTER: Locherer C
PROVIDER: S-EPMC9326634 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3'-FADP or CoA and fission yeast Rai1 with 3'-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.
| S-EPMC7293010 | biostudies-literature
Project description:SarA, a transcriptional regulator of Staphylococcus aureus, is a major global regulatory system that coordinates the expression of target genes involved in its pathogenicity. Various studies have identified a large number of SarA target genes, but an in-depth characterization of the sarA regulon, including small regulatory RNAs (sRNAs), has not yet been done. In this study, we utilized transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) to determine a comprehensive list of SarA-regulated targets, including both mRNAs and sRNAs. RNA-Seq analysis indicated 390 mRNAs and 51 sRNAs differentially expressed in a ΔsarA mutant, while ChIP-Seq revealed 354 mRNAs and 55 sRNA targets in the S. aureus genome. We confirmed the authenticity of several novel SarA targets by Northern blotting and electrophoretic mobility shift assays. Among them, we characterized repression of sprG2, a gene that encodes the toxin of a type I toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2. Finally, a novel SarA consensus DNA binding sequence was generated using the upstream promoter sequences of 15 novel SarA-regulated sRNA targets. A genome-wide scan with a deduced SarA motif enabled the discovery of new potential SarA target genes which were not identified in our RNA-Seq and ChIP-Seq analyses. The strength of this new consensus was confirmed with one predicted sRNA target. The RNA-Seq and ChIP-Seq combinatory analysis gives a snapshot of the regulation, whereas bioinformatic analysis reveals a permanent view of targets based on sequence. Altogether these experimental and in silico methodologies are effective to characterize transcriptional factor (TF) regulons and functions. IMPORTANCE Staphylococcus aureus, a commensal and opportunist pathogen, is responsible for a large number of human and animal infections, from benign to severe. Gene expression adaptation during infection requires a complex network of regulators, including transcriptional factors (TF) and sRNAs. TF SarA influences virulence, metabolism, biofilm formation, and resistance to some antibiotics. SarA directly regulates expression of around 20 mRNAs and a few sRNAs. Here, we combined high-throughput expression screening methods combined with binding assays and bioinformatics for an in-depth investigation of the SarA regulon. This combinatory approach allowed the identification of 85 unprecedented mRNAs and sRNAs targets, with at least 14 being primary. Among novel SarA direct targets, we characterized repression of sprG2, a gene that encodes the toxin of a toxin-antitoxin system, indicating a multilayer lockdown of toxin expression by both SarA and its cognate antitoxin, SprF2.
| S-EPMC8510525 | biostudies-literature
Project description:Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon Thermococcus kodakarensis encodes two homologs of bacterial DPCK and the DPCK domain of eukaryotic CoA synthase, TK1334 and TK2192. We purified the recombinant TK1334 and TK2192 proteins and found that they lacked DPCK activity. Bioinformatic analyses showed that, in several archaea, the uncharacterized gene from arCOG04076 protein is fused with the gene for phosphopantetheine adenylyltransferase (PPAT), which catalyzes the reaction upstream of the DPCK reaction in CoA biosynthesis. This observation suggested that members of arCOG04076, both fused to PPAT and standalone, could be the missing archaeal DPCKs. We purified the recombinant TK1697 protein, a standalone member of arCOG04076 from T. kodakarensis, and demonstrated its GTP-dependent DPCK activity. Disruption of the TK1697 resulted in CoA auxotrophy, indicating that TK1697 encodes a DPCK that contributes to CoA biosynthesis in T. kodakarensis TK1697 homologs are widely distributed in archaea, suggesting that the arCOG04076 protein represents a novel family of DPCK that is not homologous to bacterial and eukaryotic DPCKs but is distantly related to bacterial and eukaryotic thiamine pyrophosphokinases. We also constructed and characterized gene disruption strains of TK0517 and TK2128, homologs of bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and PPAT, respectively. Both strains displayed CoA auxotrophy, indicating their contribution to CoA biosynthesis. Taken together with previous studies, the results experimentally validate the entire CoA biosynthesis pathway in T. kodakarensisIMPORTANCE CoA is utilized in a wide range of metabolic pathways, and its biosynthesis is essential for all life. Pathways for CoA biosynthesis in bacteria and eukaryotes have been established. In archaea, however, the enzyme that catalyzes the final step in CoA biosynthesis, dephospho-CoA kinase (DPCK), had not been identified. In the present study, bioinformatic analyses identified a candidate for the DPCK in archaea, which was biochemically and genetically confirmed in the hyperthermophilic archaeon Thermococcus kodakarensis Genetic analyses on genes presumed to encode bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and phosphopantetheine adenylyltransferase confirmed their involvement in CoA biosynthesis. Taken together with previous studies, the results reveal the entire pathway for CoA biosynthesis in a single archaeon and provide insight into the different mechanisms of CoA biosynthesis and their distribution in nature.
| S-EPMC6650551 | biostudies-literature
Project description: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.
| S-EPMC2965222 | biostudies-literature
Project description: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) 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 also 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.
| S-EPMC2790875 | biostudies-literature
Project description:Pseudomonas aeruginosa and Staphylococcus aureus mixed-species biofilm infections are more resilient to biocide attacks compared to their single-species counterparts. Therefore, this study used an in vitro model recapitulating bacterial burdens seen in in vivo infections to investigate the interactions of P. aeruginosa and S. aureus in biofilms. RNA sequencing (RNA-seq) was utilized to identify the entire genomic response, both open reading frames (ORFs) and small RNAs (sRNAs), of each species. Using competitive indexes, transposon mutants validated uncharacterized PA1595 of P. aeruginosa and Panton-Valentine leukocidin ORFs of S. aureus are required for competitive success. Assessing spent media on biofilm development determined that the effects of these ORFs are not solely mediated by mechanisms of secretion. Unlike PA1595, leukocidin (lukS-PV) mutants of S. aureus lack a competitive advantage through contact-mediated mechanisms demonstrated by cross-hatch assays. RNA-seq results suggested that during planktonic mixed-species growth there is a robust genomic response or active combat from both pathogens until a state of equilibrium is reached during the maturation of a biofilm. In mixed-species biofilms, P. aeruginosa differentially expressed only 0.3% of its genome, with most ORFs necessary for growth and biofilm development, whereas S. aureus modulated approximately 5% of its genome, with ORFs suggestive of a phenotype of increased virulence and metabolic quiescence. Specific expression of characterized sRNAs aligned with the genomic response to presumably coordinate the adaptive changes necessary for this homeostatic mixed-species biofilm and sRNAs may provide viable foci for the design of future therapeutics.
| S-EPMC5458535 | biostudies-literature
Project description:The wide variety of protein structures and functions results from the diverse properties of the 20 canonical amino acids. The generally accepted hypothesis is that early protein evolution was associated with enrichment of a primordial alphabet, thereby enabling increased protein catalytic efficiencies and functional diversification. Aromatic amino acids were likely among the last additions to genetic code. The main objective of this study was to test whether enzyme catalysis can occur without the aromatic residues (aromatics) by studying the structure and function of dephospho-CoA kinase (DPCK) following aromatic residue depletion. We designed two variants of a putative DPCK from Aquifex aeolicus by substituting (a) Tyr, Phe and Trp or (b) all aromatics (including His). Their structural characterization indicates that substituting the aromatics does not markedly alter their secondary structures but does significantly loosen their side chain packing and increase their sizes. Both variants still possess ATPase activity, although with 150-300 times lower efficiency in comparison with the wild-type phosphotransferase activity. The transfer of the phosphate group to the dephospho-CoA substrate becomes heavily uncoupled and only the His-containing variant is still able to perform the phosphotransferase reaction. These data support the hypothesis that proteins in the early stages of life could support catalytic activities, albeit with low efficiencies. An observed significant contraction upon ligand binding is likely important for appropriate organization of the active site. Formation of firm hydrophobic cores, which enable the assembly of stably structured active sites, is suggested to provide a selective advantage for adding the aromatic residues.
| S-EPMC8040869 | biostudies-literature
Project description:Staphylococcus aureus is a commensal bacterium and pathogen. Identifying biomarkers for the transition from colonization to disease caused by this organism would be useful. Several S. aureus small RNAs (sRNAs) regulate virulence. We investigated presence and expression of 8 sRNAs in 83 S. aureus strains from 42 patients with sepsis or septic shock and 41 asymptomatic colonized carriers. Small pathogenicity island sRNAs sprB and sprC were clade specific. Six sRNAs had variable expression not correlated with clinical status. Expression of RNAIII was lower in strains from septic shock patients than in strains from colonized patients. When RNAIII was associated with expression of sprD, colonizing strains could be discriminated from strains in patients with bloodstream infections, including patients with sepsis and septic shock. Isolates associated with colonization might have sRNAs with target expression different from those of disease isolates. Monitoring expression of RNAIII and sprD could help determine severity of bloodstream infections.
| S-EPMC4994353 | biostudies-literature
Project description:Dephospho-CoA kinases (DPCKs) are members of the kinase family that catalyze the final step in CoA biosynthesis. Their function is phosphorylation of the 3-hydroxyl group of the ribose using ATP as a phosphate donor. Structural changes induced by ATP binding play an important role during the DPCK catalytic cycle. In this work, DPCK from Legionella pneumophila was overexpressed in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are described. The protein was crystallized in space group P21212, with unit-cell parameters a = 36.29, b = 82.20, c = 81.80 Å, using the hanging-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method.
| S-EPMC4014329 | biostudies-literature
Project description:Small RNAs (sRNAs) are involved in the post-transcriptional regulation of metabolic pathways and in responses to stress and virulence. We analyzed the expression levels of five sRNAs of Staphylococcus aureus during human colonization or infection. Total RNA was isolated from nasal carriers, abscesses and cystic fibrosis patients (20 subjects per condition). The expression levels of the sRNAs were measured in the clinical samples and compared with those of the corresponding strains grown in vitro. Five sRNAs were encoded and expressed in all clinical strains in vitro. In vivo, the global expression of the five sRNAs was extremely variable in the abscessed patients, more homogeneous in the cystic fibrosis patients, and highly uniform in the nasal carrier samples. The expression levels of the sRNAs in vivo resembled those obtained at exponential phase or late exponential phase of growth in vitro, for three and one sRNA respectively; while for one sRNA, the expression was always higher in vivo as compared to in vitro growth. The in vitro conditions do not uniformly mimic the in vivo conditions for sRNA expression. Nasal colonization is associated with a unique expression pattern of sRNA that might reflect the commensalism of S. aureus in this niche.
| S-EPMC3358344 | biostudies-other