Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Identification and application of a panel of constitutive promoters for gene overexpression in Staphylococcus aureus

ABSTRACT: Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-E. coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of egfp gene under the control of different promoters was confirmed and quantified by Western-blot and qPCR, which suggested that the activity of these promoters varied from 18% to 650% of the activity of PsarA, a widely used promoter for gene expression currently. In addition, our constructed vectors were verified to be of high compatibility on gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used in overexpressing two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (purR). Meanwhile, the physiological functions and phenotypes of overexpressed purR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This developed system will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE189644 | GEO | 2021/11/30

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Stress-induced inactivation of the Staphylococcus aureus purine biosynthesis repressor leads to hypervirulence

Project description:Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.

2019-02-26 | GSE124869 | GEO

RNA-Seq of WT Staphylococcus aureus and dpurR grown to exponential and stationary phase

Project description:We report here that mutants for purR display altered transcriptional profiles compared to WT S. aureus at multiple stages of growth.

2019-06-12 | GSE132179 | GEO

Project description:Catalase Protects Biofilm of Staphylococcus aureus against Daptomycin Activity

| PRJNA725634 | ENA

A systematic and scalable approach for dissecting the molecular 2 mechanisms of transcriptional regulation in bacteria

Project description:Gene regulation is one of the most ubiquitous processes in biology. And yet, while the catalogue of 15 bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in 16 these genomes are regulated. Characterizing the molecular mechanisms by which regulatory sequences 17 operate still requires focused efforts using low-throughput methods. Here we show how a combination of 18 massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used 19 to dissect bacterial promoters in a systematic and scalable way. We demonstrate this method on both 20 well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all 21 cases we recover nucleotide-resolution models of promoter mechanism. For some promoters, including 22 previously unannotated ones, we can further extract quantitative biophysical models describing 23 input-output relationships. This method opens up the possibility of exhaustively dissecting the 24 mechanisms of promoter function in E. coli and a wide range of other bacteria.

2018-04-13 | PXD007892 | JPOST Repository

Characterization of alternative promoter usage in advanced prostate cancer

Project description:In this study, we use deep whole genome transcriptome sequencing to show promoter activity changes during progression from normal prostate cells to advanced prostate disease. We show how androgen signaling and somatic alterations such as loss of the tumor suppressor RB1 or the activation of the ERG oncogene are linked to activity of transcription factors at noncanonical promoters. Alternative promoter usage analysis more accurately reflects changes in TF network during disease progression and in response to therapy. Lastly, we use matched whole genome methylation data to demonstrate a link between methylation and alternative promoter activity, dramatically improving the strength of association between gene expression and methylation data.

| EGAS00001006275 | EGA

High-throughput functional comparison of promoter and enhancer activities

Project description:Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated co-activator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, while others are not. While the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators.

2016-05-23 | GSE77213 | GEO

Steady-state hydrogen peroxide induces glycolysis via metabolic reroute in P. aeruginosa and S. aureus

Project description:GAPDHs from human pathogens S. aureus and P. aeruginosa can be readily inhibited by ROS-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 induces rapid metabolic reroute from glycolysis to pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces a metabolic reroute from glycolysis to PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Given that H2O2 can be produced constitutively by the host immune response, exposure to the steady-state stress of H2O2 recapitulates more accurately bacterial responses to host immune system in vivo. RNA-seq in Pseudomonas aeruginosa and Staphylococus aureus under steady state of H2O2

2014-05-08 | E-GEOD-55528 | biostudies-arrayexpress

Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells

Project description:Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with VSV-G were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The short elongation factor 1α (EFS) promoter mediated the most robust expression, while expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared to all other constructs tested here. The function of UCOE diminished but did not fully counteract vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.

2018-01-01 | GSE94157 | GEO

The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus

Project description:The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes from soil bacteria to pathogens: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis. We identified PurR as a receptor of (p)ppGpp in Bacillus anthracis and revealed that (p)ppGpp strongly enhances PurR binding to its regulon in the Bacillus subtilis genome. A co-structure reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of PRT enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant fails to downregulate purine synthesis genes upon starvation. Our work establishes the precedent of (p)ppGpp as a classical effector of transcription repressors and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from the human intestinal tract to host-pathogen interfaces.

2021-12-10 | GSE185164 | GEO

Genome-wide mapping of autonomous promoter activity in human cells

Project description:Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of sequences that could be tested. Here we present Survey of Regulatory Elements (SuRE), a method to assay more than 10^8 DNA fragments, each 0.2-2kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library is constructed of random genomic fragments upstream of a 20bp barcode and decoded by paired-end sequencing. This library is then transfected into cells and transcribed barcodes are quantified in the RNA by high throughput sequencing. When applied to the human genome, we achieved a 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide. By computational modeling we delineated subregions within promoters that are relevant for their activity. For instance, we show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.

2016-11-10 | GSE78709 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data