Expression profiling of B.subtils SMY wild-type vs. (p)ppGpp0 cells upon amino acid starvation.
Ontology highlight
ABSTRACT:
ORGANISM(S): Bacillus subtilis
PROVIDER: GSE39758 | GEO | 2012/11/01
SECONDARY ACCESSION(S): PRJNA171588
REPOSITORIES: GEO
Similar Datasets
Project description:Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a post-transcriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival entirely by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction. Four-condition experiment: wt, wt+RHX, (p)ppGpp0, (p)ppGpp0+RHX. Biological replicates: 3 for each sample. Reference: a mixture of wt RNA from different growth phases and wt backgrounds.
2012-11-01 | E-GEOD-39758 | biostudies-arrayexpress
Project description:The nucleotide (p)ppGpp is crucial for viability during amino acid limitation in bacteria, yet how it accomplishes this remains unknown. We found that the absence of (p)ppGpp in Bacillus subtilis cells leads to multiple amino acid auxotrophy, and that (p)ppGpp allows for prototrophy by reducing GTP levels. We provide evidence that reduction of GTP levels relieves the requirements for branched-chain amino acids primarily by preventing hyperactivity of the GTP-dependent transcriptional regulator CodY, but that GTP levels can also play an important role in regulating transcription of many amino acid biosynthesis genes independently of CodY. Thus, CodY-dependent and independent regulation of transcription by GTP levels plays overlapping yet distinct physiological roles in allowing amino acid prototrophy. Finally, supplementing these required amino acids does not protect against cell death upon nutrient downshift, but allows for sustained growth following this transition. We conclude that regulation of GTP levels by (p)ppGpp allows cells to adapt to conditions of amino acid limitation by first allowing survival during shifting nutrient conditions, and then allowing amino acid prototrophy by transcriptionally regulating amino acid biosynthesis. This strategy may be used to ensure viability during amino acid limitation in evolutionarily divergent bacteria.
2013-11-01 | GSE41501 | GEO
Project description:The nucleotide (p)ppGpp is crucial for viability during amino acid limitation in bacteria, yet how it accomplishes this remains unknown. We found that the absence of (p)ppGpp in Bacillus subtilis cells leads to multiple amino acid auxotrophy, and that (p)ppGpp allows for prototrophy by reducing GTP levels. We provide evidence that reduction of GTP levels relieves the requirements for branched-chain amino acids primarily by preventing hyperactivity of the GTP-dependent transcriptional regulator CodY, but that GTP levels can also play an important role in regulating transcription of many amino acid biosynthesis genes independently of CodY. Thus, CodY-dependent and independent regulation of transcription by GTP levels plays overlapping yet distinct physiological roles in allowing amino acid prototrophy. Finally, supplementing these required amino acids does not protect against cell death upon nutrient downshift, but allows for sustained growth following this transition. We conclude that regulation of GTP levels by (p)ppGpp allows cells to adapt to conditions of amino acid limitation by first allowing survival during shifting nutrient conditions, and then allowing amino acid prototrophy by transcriptionally regulating amino acid biosynthesis. This strategy may be used to ensure viability during amino acid limitation in evolutionarily divergent bacteria. Twelve-condition experiment: wt, wt+RHX, wt+Guo, (p)ppGpp0, (p)ppGpp0+RHX, (p)ppGpp0+Guo, M-NM-^TcodY (p)ppGpp0, M-NM-^TcodY (p)ppGpp0+RHX, M-NM-^TcodY (p)ppGpp0+Guo, guaB- (p)ppGpp0, guaB- (p)ppGpp0+RHX, guaB- (p)ppGpp0+Guo. Biological replicates: 3 for each sample. Reference: a mixture of wt RNA from different growth phases and wt backgrounds.
2013-11-01 | E-GEOD-41501 | biostudies-arrayexpress
Project description:The production of (p)ppGpp by Streptococcus mutans UA159 is catalyzed by three gene products, RelA, RelP and RelQ. Here, we investigate the role of the RelA (Rel) homologue of S. mutans in the stringent response and in global control of gene expression. RelA of S. mutans was shown to synthesize pppGpp in vitro from GTP and ATP in the absence of added ribosomes, as well as in vivo in an E. coli relA-spoT mutant. Mupirocin (MUP) was shown to induce high levels of (p)ppGpp production in S. mutans in a relA-dependent manner, with a concommitant reduction in GTP pools. Keywords: (p)ppGpp, nutrient starvation, biofilm, virulence, stress
2007-11-14 | GSE9382 | GEO
Project description:Transcriptional profiles of wildtype and mutant strains bt0700 and bt0700 bt3998 (ppGpp0) of human gut commensal B. thetaiotaomicron were collected to investigate the relevance of the stringent response mediated by the alarmone (p)ppGpp for growth and survival in three different conditions. RNAseq samples were taken during growth in glucose minimal medium, after 1h of carbon starvation, and after 6 days of monocolonizing germ-free mice (from cecum material).
2018-07-04 | E-MTAB-6412 | biostudies-arrayexpress
Project description:The alarmone species ppGpp and pppGpp are an important part of bacterial physiology as they both coordinate the bacterial stress response to nutrient deficiency and also contribute to the maintenance of cell homeostasis. Recently, all components of the (p)ppGpp metabolism for the industrially relevant actinobacterial model organism Corynebacterium glutamicum could be functionally characterized by in vitro analysis and heterologous expression in the model system Escherichia coli. Based on these results, the strain C. glutamicum CR099 ΔrelΔrelSΔrelH is the first C. glutamicum (p)ppGpp0 strain available. The aim of this study was to investigate the effects of the alarmone species on transcriptome regulation in C. glutamicum by differential transcriptome analysis of the parental strain and the (p)ppGpp0 mutant. An RNAseq analysis of the early exponential growth phase was performed to analyze the (p)ppGpp basal level effects (Illumina TruSeq stranded mRNA libraries sequenced on Illumina HiSeq rapid mode 2 x 25nt PE). Due to the direct association of the alarmones with the bacterial stress response, the transcriptional response to the induction of a total starvation situation was investigated in form of a time series. Following an analysis of pooled replicates for 5 time points (Illumina TruSeq stranded mRNA libraries sequenced on Illumina HiSeq rapid mode 2 x 50nt PE), a detailed investigation was carried out for two stress exposure durations using three biological replicates per strain and time point each (Illumina TruSeq stranded mRNA libraries sequenced on Illumina HiSeq rapid mode 2 x 25nt PE).
2019-12-02 | E-MTAB-8070 | biostudies-arrayexpress
Project description:Guanosine 3 ,5 -bispyrophosphate (ppGpp), also known as ‘‘magic spot,’’ has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wildtype Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. When cultured on a mixture of glucose and lactose, E. coli grows preferentially on glucose until it is exhausted, resulting in growth arrest while the cells adjust to growth on lactose, i.e., diauxie. Diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine.
2007-03-17 | GSE7265 | GEO
Project description:Guanosine 3 ,5 -bispyrophosphate (ppGpp), also known as ‘‘magic spot,’’ has been shown to bind prokaryotic RNA polymerase to down-regulate ribosome production and increase transcription of amino acid biosynthesis genes during the stringent response to amino acid starvation. Because many environmental growth perturbations cause ppGpp to accumulate, we hypothesize ppGpp to have an overarching role in regulating the genetic program that coordinates transitions between logarithmic growth (feast) and growth arrest (famine). We used the classic glucose-lactose diauxie as an experimental system to investigate the temporal changes in transcription that accompany growth arrest and recovery in wildtype Escherichia coli and in mutants that lack RelA (ppGpp synthetase) and other global regulators, i.e., RpoS and Crp. When cultured on a mixture of glucose and lactose, E. coli grows preferentially on glucose until it is exhausted, resulting in growth arrest while the cells adjust to growth on lactose, i.e., diauxie. Diauxie was delayed in the relA mutant and was accompanied by a 15% decrease in the number of carbon sources used and a 3-fold overall decrease in the induction of RpoS and Crp regulon genes. Thus the data significantly expand the previously known role of ppGpp and support a model wherein the ppGpp-dependent redistribution of RNA polymerase across the genome is the driving force behind control of the stringent response, general stress response, and starvation-induced carbon scavenging. Our conceptual model of diauxie describes these global control circuits as dynamic, interconnected, and dependent upon ppGpp for the efficient temporal coordination of gene expression that programs the cell for transitions between feast and famine. E. coli MG1655 and isogenic mutants were cultured in a 2 l Biostat B fermentor (B. Braun Biotech International) containing 1 liter of Morpholinepropanesulfonic acid (MOPS) minimal medium with 0.5 g/l of glucose and 1.5 g/l of lactose. The temperature was maintained at 37 ºC and pH was kept constant at 7.2 by the addition of 2 M NaOH. The dissolved oxygen level was maintained above 20% of saturation by adjusting the agitation speeds in the range of 270-500 rpm with fixed 1 l/min air flow.
2007-03-17 | E-GEOD-7265 | biostudies-arrayexpress
Project description:Transcription profiling of wild type, relA-, and relA-spoT-, crp-, dksA-, rpoS-, lrp- mutant strains of E. coli starved for isoleucine Bacteria comprehensively reorganize their global gene expression when faced with nutrient exhaustion. In Escherichia coli and other free-living bacteria, the alarmone ppGpp facilitates this massive response by directly or indirectly coordinating the down-regulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation likely requires the cooperative activities of many different genetic regulators, yet the structure of the transcription network below the level of ppGpp remains poorly defined. Using isoleucine starvation as an experimental model system for amino acid starvation, we identified genes that required ppGpp, Lrp, and RpoS for their induction. Surprisingly, despite the fact that the overwhelming majority of genes controlled by Lrp and RpoS required ppGpp for their activation, we found that these two regulons were not induced simultaneously. The data reported here suggest that metabolic genes, such as those of the Lrp regulon, require only a low basal level of ppGpp for their efficient induction. In contrast, the RpoS-dependent general stress response is not robustly induced until relatively high levels of ppGpp accumulate. Here we describe a data-driven conceptual model that explains how bacterial cells allocate transcriptional resources between metabolic and stress survival processes by discretely tuning regulatory activities to a central indicator of cellular physiology. The regulatory structure that emerges is consistent with a rheostatic model of the stringent response that allows cells to efficiently adapt to a wide range of nutritional environments. Keywords: genetic modification design; stress response; isoleucine starvation
2008-04-08 | GSE11087 | GEO
Project description:The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rshSyn, codY and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rshSyn and rshSyn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psma1-4 and/or psmM-CM-^_1,2 could complement the survival of the rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis. Microarray was used to evaluate alteration in the transcriptome of codY and rsh mutants and compared to the wild type
2013-04-05 | E-GEOD-45617 | biostudies-arrayexpress