Dataset Information

Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5'-Caps, but No CoAlation Marks.

ABSTRACT: Novel features of coenzyme A (CoA) and its precursor, 3'-dephospho-CoA (dpCoA), recently became evident. dpCoA was found to attach to 5'-ends of small ribonucleic acids (dpCoA-RNAs) in two bacterial species (Escherichia coli and Streptomyces venezuelae). Furthermore, CoA serves, in addition to its well-established coenzymatic roles, as a ubiquitous posttranslational protein modification ('CoAlation'), thought to prevent the irreversible oxidation of cysteines. Here, we first identified and quantified dpCoA-RNAs in the small RNA fraction of the human pathogen Staphylococcus aureus, using a newly developed enzymatic assay. We found that the amount of dpCoA caps was similar to that of the other two bacteria. We furthermore tested the hypothesis that, in the environment of a cell, the free thiol of the dpCoA-RNAs, as well as other sulfur-containing RNA modifications, may be oxidized by disulfide bond formation, e.g., with CoA. While we could not find evidence for such an 'RNA CoAlation', we observed that CoA disulfide reductase, the enzyme responsible for reducing CoA homodisulfides in S. aureus, did efficiently reduce several synthetic dpCoA-RNA disulfides to dpCoA-RNAs in vitro. This activity may imply a role in reversing RNA CoAlation.

SUBMITTER: Locherer C

PROVIDER: S-EPMC9326634 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5'-Caps, but No CoAlation Marks.

Löcherer Christian C Bühler Nadja N Lafrenz Pascal P Jäschke Andres A

Non-coding RNA 20220628 4

Novel features of coenzyme A (CoA) and its precursor, 3'-dephospho-CoA (dpCoA), recently became evident. dpCoA was found to attach to 5'-ends of small ribonucleic acids (dpCoA-RNAs) in two bacterial species (Escherichia coli and Streptomyces venezuelae). Furthermore, CoA serves, in addition to its well-established coenzymatic roles, as a ubiquitous posttranslational protein modification ('CoAlation'), thought to prevent the irreversible oxidation of cysteines. Here, we first identi ...[more]

PMID: 35893229

Similar Datasets

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.

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.

Dataset Information

Staphylococcus aureus Small RNAs Possess Dephospho-CoA 5'-Caps, but No CoAlation Marks.

Publications

<i>Staphylococcus aureus</i> Small RNAs Possess Dephospho-CoA 5'-Caps, but No CoAlation Marks.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets