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: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
Project description:The pathogenic spirochete Borrelia burgdorferi senses and responds to diverse environmental challenges, including changes in nutrient availability, throughout its natural infectious cycle in Ixodes spp. ticks and mammalian hosts. This study examined the role of the putative DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA-deficient B. burgdorferi strains were subjected to starvation by shifting mid-logarithmic cultures grown in BSK-II medium to serum-free RPMI medium for six hours under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi as the dksA-deficient B. burgdorferi strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed by wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFUs were observed for dksA-deficient spirochetes after prolonged starvation in RPMI medium compared to wild-type B. burgdorferi. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthase RelBbu, while the DksA regulon also included many plasmid-borne genes. Corresponding to a DksA-(p)ppGpp regulatory relationship, (p)ppGpp levels were constitutively elevated in the dksA-deficient strain compared to the wild-type strain. Together, these data indicate that DksA directs the stringent response with a regulatory interplay with (p)ppGpp that is fundamental to B. burgdorferi responses to the environment.
2018-12-18 | GSE119023 | GEO
Project description:Polyphosphate (polyP) is a ubiquitous and abundant compound found in bacteria, fungi, algae, plant, and animals. Among roles of intracellular polyP in bacteria are resistance and survival in the stationary phase of the growth against stress and stringent condition. Therefore, intracellular polyP is considered as a virulence factor of bacteria. In contrast, exogenous polyP has an antimicrobial activity against a variety of microorganisms. To date, much has been numerous studies of antibacterial effect of polyP against gram positive bacteria and fungi while relatively little reports have been published concerning gram negative bacteria. Here we describe bactericidal effect of polyP against gram negative periodontopathic bacterium, Porphyromonas gingivalis, and the transcriptional change by polyP in this bacterium. The bacterial growth was inhibited by polyP (chain length of P3~P75) at concentration of 0.02~0.03%, but not by Pi and PPi at the concentrations. polyP75 was chosen for further experiments, which suppressed the further growth of P. gingivalis as low as 0.03%. polyP75 completely killed the bacterial cells at the concentration of 0.035%. Microarray analysis was employed to identify genes that showed a greater than 1.5-fold difference in the expression by polyP75 at the concentration of 0.03%. It was found that 155 genes were up-regulated and 173 were down-regulated. Down-regulated genes include groups of energy metabolism-related genes, cell envelope-related genes, and genes in relation to biosynthesis of cofactors, prosthetic groups and carriers. Among the down-regulated genes were several involved in DNA replication, cell division protein, and biosynthesis of purines, pyrimidines, nucleosides and nucleotides. In contrast, a large number of ribosomal proteins, transciptional regulators and transposases were up-regulated. Oxidative stress-related genes and iron storage proteins also appeared to be increased in the expression. Real-time PCR confirmed up- and down-regulation of some selected genes. The overall results suggest that polyP has a bactericidal activity against P. gingivalis, interfering with translation, energy metabolism, DNA replication, cell division, and biosynthesis of purines, pyrimidines, nucleoside and nucleotides. polyP may be used as an agent for prevention and treatment of periodontal infections. Polyp-treated vs. untreated intensity ratio data linked below as a supplementary file. Keywords: agent response
2008-05-30 | GSE11471 | GEO