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Expression profiling of B.subtils SMY wild-type vs. (p)ppGpp0 cells upon amino acid starvation.

ABSTRACT: 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.

ORGANISM(S): Bacillus subtilis

SUBMITTER: Jue Wang

PROVIDER: E-GEOD-39758 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Expression profiling of B.subtils SMY wild-type, (p)ppGpp0, M-NM-^TcodY (p)ppGpp0 and guaB- (p)ppGpp0 cells upon amino acid starvation (RHX) or guanosine (Guo) treatment.

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

Expression profiling of B.subtils SMY wild-type vs. (p)ppGpp0 cells upon amino acid starvation.

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.

2012-11-01 | GSE39758 | GEO

Expression profiling of B.subtils SMY wild-type, (p)ppGpp0, ΔcodY (p)ppGpp0 and guaB- (p)ppGpp0 cells upon amino acid starvation (RHX) or guanosine (Guo) treatment.

2013-11-01 | GSE41501 | GEO

RNAseq of the human gut commensal Bacteroides thetaiotaomicron wildtype, bt0700 and bt0700 bt3998 (ppGpp0) strains across three conditions: glucose minimal medium, 1h of carbon starvation, and mouse gut colonization

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

Transcriptional analysis of regulatory effects in Corynebacterium glutamicum and a derived (p)ppGpp0 mutant strain

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

Expression data from E. coli cells overexpressing either GreA or GreB in ppGpp0 cells in the dksA+ or dksA- background

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

Expression data from E. coli cells overexpressing either GreA or GreB in ppGpp0 cells in the dksA+ or dksA- background

2011-10-27 | GSE28795 | GEO

RelA/(p)ppGpp regulates the glucose-starvation induced stringent response in the zoonostic Streptococcus suis

Project description:Growth curves and (p)ppGpp accumulation assays showed that RelA inactivation could influence S. suis growth and led to incapacity of (p)ppGpp synthesis during glucose starvation. To identify the roles of RelA/(p)ppGpp in global gene regulation in S. suis, we compared the transcriptional profiles of SC-19 [a (p)ppGpp+ strain] and ΔrelA [a (p)ppGpp0 strain during glucose starvation] in both glucose-abundant and -deficient CDM in exponential phase by microarray analysis. A less stringent cut-off limit, 2-fold change, was used. qRT-PCR validation displayed the same trends observed in the microarrays

2015-08-08 | GSE70092 | GEO

RNAseq of Staphylococcus aureus wildtype and ppGpp0 mutant reveals rolen of alarmon (p)ppGpp confering tolerance to oxidative stress and antibiotics during the stationary phase in Staphylococcus aureus

Project description:RNAseq of coding RNA in Staphylococcus aureus wildtype and ppGpp0 mutant shows transcription of the Fe-storage ferritin (ftnA) and miniferritin (dps) was elevated in the ppGpp0 mutant, while Fur-regulated iron transporters for uptake of heme or siderophores were repressed, including the isdABCDEFG, sirABC and sstADBCD-operons. Together these results indicate that (p)ppGpp confers tolerance to ROS and antibiotics during the stationary phase by down-regulation of internal iron levels to prevent ROS formation.

2020-08-31 | E-MTAB-9368 | biostudies-arrayexpress

The Role of RelA of Streptococcus mutans in Global Control of Gene Expression

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

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