Understanding the roles of dksA and greA in inorganic polyphosphate accumulation in Escherichia coli
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE144816 | GEO | 2020/03/11
REPOSITORIES: GEO
Similar Datasets
Project description:Understanding the roles of dksA and greA in inorganic polyphosphate accumulation in Escherichia coli
| PRJNA604995 | ENA
Project description:Strains devoid of ppGpp (ΔrelA ΔspoT; called ppGpp0), and ppGpp0 dksA- exhibit several amino acid requirements for growth on minimal media. We found that overexpression of DksA can complement some of those requirements. Since DksA is a factor that binds to the RNA polymerase secondary channel, we wondered if other secondary channel proteins might also exert a similar role with respect to growth on minimal media. In our study we found that GreA and partially GreB can in fact complement these requirements under certain conditions. Here, we wished to investigate a broader effect of GreA and GreB on ppGpp0 and ppGpp0 dksA- strains. Since the parent strains are unable to grow in minimal media, we had to supplement the M9 glucose medium with a set of amino acids (DFHILQSTV). We found that both, GreA and GreB can affect a much larger set of genes in the absence of dksA, than in its presence. Also, GreA seems to affect more genes than GreB, under both conditions. We used microarrays to detail the effects of overproducing either GreA or GreB in cells devoid of ppGpp, in the dksA+ or dksA- background E. coli cells harbouring plasmids carrying either greA (pA = pHM1873) or greB (pB = pHM1874) genes, or vector control (pGB2), were monitored in an effort to elucidate the effects of their protein products. This was done in two backgrounds- either in E. coli ppGpp0 cells (ΔrelA, ΔspoT) carrying wt dksA gene or a dksA deletion.
2011-10-26 | E-GEOD-28795 | biostudies-arrayexpress
Project description:Inorganic polyphosphate (Poly P) is a polymer of various phosphate residues linked by phosphoanhydride bonds as in ATP. It is found in all cells in nature with roles in the origin and survival of species, particularly in bacteria. To study the role of the inorganic polyphosphate in bacteria, we obtained knockout mutants of polyP metabolism genes in Escherichia coli K12. We performed DNA microarray experiments of single mutants in polyphosphate kinase 1 (PPK1), exopolyphosphatase (PPX) and also with the double mutant (PPK1 and PPX). The mutant strains growth normally in LB medium but have different colony morphology phenotypes. All mutants have flagellation problems and a detail description of all gain and lost phenotypes o these strains will be published soon because we performed a complete phenotypic microarray study of all three mutant strains.
2011-06-15 | GSE29954 | GEO
Project description:Strains devoid of ppGpp (ΔrelA ΔspoT; called ppGpp0), and ppGpp0 dksA- exhibit several amino acid requirements for growth on minimal media. We found that overexpression of DksA can complement some of those requirements. Since DksA is a factor that binds to the RNA polymerase secondary channel, we wondered if other secondary channel proteins might also exert a similar role with respect to growth on minimal media. In our study we found that GreA and partially GreB can in fact complement these requirements under certain conditions. Here, we wished to investigate a broader effect of GreA and GreB on ppGpp0 and ppGpp0 dksA- strains. Since the parent strains are unable to grow in minimal media, we had to supplement the M9 glucose medium with a set of amino acids (DFHILQSTV). We found that both, GreA and GreB can affect a much larger set of genes in the absence of dksA, than in its presence. Also, GreA seems to affect more genes than GreB, under both conditions. We used microarrays to detail the effects of overproducing either GreA or GreB in cells devoid of ppGpp, in the dksA+ or dksA- background
2011-10-27 | GSE28795 | GEO
Project description:Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA damage response and recruits the main recombination protein RecA. This microarray experiment compares the transcriptional responses to amino acid starvation in wild-type and delta dksA cells. The SOS-regulated genes are highly induced in delta dksA cells.
2010-06-01 | GSE19742 | GEO
Project description:The emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains poses a significant threat to public health due to their high mortality rates and propensity to cause severe community-acquired infections in otherwise healthy individuals. The ability of hvKP to form biofilms and produce a protective capsule contributes to its enhanced virulence and is a significant challenge to effective antibiotic treatment. Therefore, understanding the molecular mechanisms underlying hvKP virulence and biofilm formation is crucial for developing new therapeutic strategies. Polyphosphate Kinase 1 (PPK1) is an enzyme responsible for inorganic polyphosphate synthesis and plays a vital role in regulating various physiological processes in bacteria. In this study, we investigated the impact of polyP metabolism on the biofilm and capsule formation and virulence traits in hvKP using Dictyostelium discoideum amoeba as a model host. We found that the PPK1 null-mutant was impaired in biofilm and capsule formation and showed attenuated virulence in D. discoideum compared to the wild-type strain. We performed a shotgun proteomic analysis of the PPK1 mutant and wild-type strain to gain further insight into the underlying molecular mechanism. The results revealed that the PPK1 mutant had a differential expression of proteins (DEP) involved in capsule synthesis (Wzi - Ugd), biofilm formation (MrkC-D-H), synthesis of the colibactin genotoxin precursor (ClbB), as well as proteins associated with the synthesis and modification of lipid A (ArnB -LpxC - PagP). These proteomic findings corroborate the phenotypic observations and indicate that the PPK1 mutation is associated with impaired biofilm and capsule formation and attenuated virulence in hypervirulent K. pneumoniae. Overall, our study highlights the importance of polyP synthesis in regulating extracellular biomolecules and virulence in K. pneumoniae and provides insights into potential therapeutic targets for treating K. pneumoniae infections.
2024-08-23 | PXD046644 | Pride
Project description:The existing literature points towards the presence of robust mitochondrial mechanisms aimed at mitigating protein dyshomeostasis within the organelle. However, the precise molecular composition underlying these mechanisms remains unclear. Our data show that inorganic polyphosphate (polyP), a polymer well-conserved throughout evolution, is a component of these mechanisms. In mammals, mitochondria exhibit a significant abundance of polyP, and both our research and that of others have already highlighted its potent regulatory effect on bioenergetics. Given the intimate connection between energy metabolism and protein homeostasis, the involvement of polyP in proteostasis has also been demonstrated in several organisms. For example, polyP is a bacterial primordial chaperone, and its role in amyloidogenesis has already been established. Here, using HEK293 cells, our study reveals that the depletion of mitochondrial polyP leads to increased protein aggregation within the organelle, following stress exposure. Furthermore, mitochondrial polyP is able to bind to proteins, and these proteins differ under control and stress conditions. The depletion of mitochondrial polyP significantly affects the proteome under both control and stress conditions, while also exerting regulatory control over gene expression. Our findings suggest that mitochondrial polyP is a previously unrecognized, and potent component of mitochondrial proteostasis. The mass spectrometric raw files for this study are deposited here.
2024-06-28 | MSV000095178 | MassIVE
Project description:In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphates (polyP). These near universal polymers, ranging from 3 to thousands of phosphate moieties in length, are associated with molecular functions including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast S. cerevisiae, polyP synthesis by the membrane-bound VTC complex is coupled to its translocation into the lumen of the vacuole, a lysosome-related organelle, where it is stored at high concentrations. In contrast, ectopic expression of bacterial polyphosphate kinase, PPK, results in the toxic accumulation of polyP outside of the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases, and Msn2/Msn4 transcription factors. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in the yeast model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require co-factors unique to higher eukaryotes. Our work provides a mechanistic explanation for why polyP accumulation outside of lysosome-related organelles is toxic. Further, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level.
2022-02-04 | PXD031326 | Pride
Project description:In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise, and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrackblocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Chromatin immunoprecipitation (ChIP) experiments were performed by using antibodies against RNA polymerase b subunit in wild-type and DdksA cells treated with 0.5mg/ml serine hydroxamate (SHX) or untreated. DksA and s70 enrichments were compared to RNAP enrichment by ChIP experiments using antibodies against s70 and DksA in wild-type cells (also in DdksA cells as a negative control for DksA ChIP-chip). Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 19 datasets.
2014-04-21 | E-GEOD-54669 | biostudies-arrayexpress
Project description:Polyphosphates (polyP) are ancient molecules comprised of inorganic phosphates joined by high energy phosphoanhydride bonds in chains of 3-1000 units in length. In this study, we describe a system to produce polyP in mammalian cells by ectopic expression of the E. coli ppk1+ gene. Production of the EcPpk1 protein results in polyP accumulation throughout the cell, including the nucleus. Using transcriptomic and proteomic analyses, we demonstrate a broad impact of polyP on diverse pathways, including activation of the ERK1/2-EGR1 signaling axis. PolyP accumulation also results in redistribution of several chromatin bound proteins and a translation initiation factor from the nucleus to the cytoplasm. Our work will serve as a novel resource to interrogate polyP biology in higher eukaryotes.
2020-04-22 | GSE149011 | GEO