Roles of relSpn in Stringent Response, Global Regulation, and Virulence of Serotype 2 Streptococcus pneumoniae D39
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ABSTRACT:
ORGANISM(S): Streptococcus pneumoniae
PROVIDER: GSE14750 | GEO | 2010/01/11
SECONDARY ACCESSION(S): PRJNA112239
REPOSITORIES: GEO
Similar Datasets
Project description:Transcriptome analysis of D39 rel+Spn and delta-relSpn strains treated with mupirocin revealed relSpn-independent (translation stress), relSpn-dependent (stringent response), and delta-relSpn-dependent changes suggesting that relSpn and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Wildtype and rel deletion mutant (Delta rel) bacteria were grown statically in BHI broth at 37C in an atmosphere of 5% CO2. Cultures were divided in two upon reaching a density of OD620 ~0.1, and lithium mupirocin was added to one culture (t=0) to a final concentration of 100 ng per mL. Treated and untreated cultures were harvested after 20 min further incubation and RNA was extracted by a hot lysis-acid phenol protocol. We performed the following comparisons: Wildtype + mupirocin versus Wildtype untreated (reference); Delta rel + mupirocin versus Delta rel untreated (reference); Delta rel untreated versus Wildtype untreated (reference); Delta rel + mupirocin versus Wildtype + mupirocin (reference). For each comparison, microarray data were obtained from three independent biological replicates and included one dye swap.
2010-05-15 | E-GEOD-14750 | biostudies-arrayexpress
Project description:The impact on gene expression by the alarmone (p)ppGpp during growth and non-growth was determined by comparing the transcriptome of R. etli CFN42 wild type and rel mutant. This allowed us to better understand the pleiotropic stress phenotype of the rel mutant as well as identifying specific (p)ppGpp-dependent stress regulators. In order to study expression, R. etli CFN42 wild type and rel mutant was grown at 30˚C in acid minimal salts medium supplied with 10 mM NH4Cl and 10 mM succinate while monitoring the optical density (OD) of the culture. Samples were taken at OD600 = 0.3, 0.7 and 6 hours after reaching the maximum OD, representing early/late exponential and stationary phase, respectively.
2010-09-15 | E-GEOD-23961 | 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:Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and a leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by post-transcriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs have been further implicated in regulating pneumococcal pathogenesis. However, there is little overlap in the sRNAs identified among these studies, which indicates that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there are likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery's virulent S. pneumoniae strain D39 using two independent RNA-seq based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program towards identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4 including ones previously implicated in virulence are not present in strain D39 genome suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 67 new sRNA candidates in strain D39, 28 out of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112.
2019-05-13 | GSE124170 | GEO
Project description:Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen and the leading cause of bacterial pneumonia worldwide. Small regulatory RNAs (sRNAs), which often act by post-transcriptionally regulating gene expression, have been shown to be crucial for the virulence of S. pneumoniae and other bacterial pathogens. Over 170 putative sRNAs have been identified in S. pneumoniae TIGR4 strain (serotype 4) through transcriptomic studies, and a subset of these sRNAs have been further implicated in regulating pneumococcal pathogenesis. However, there was little overlap in the sRNAs identified among these studies, which indicated that the approaches used for sRNA identification were not sufficiently sensitive and robust and that there were likely many more undiscovered sRNAs encoded in the S. pneumoniae genome. Here, we sought to comprehensively identify sRNAs in Avery's virulent S. pneumoniae strain D39 using two independent RNA-seq based approaches. We developed an unbiased method for identifying novel sRNAs from bacterial RNA-seq data and have further tested the specificity of our analysis program towards identifying sRNAs encoded by both strains D39 and TIGR4. Interestingly, the genes for 15% of the putative sRNAs identified in strain TIGR4 including ones previously implicated in virulence were not present in strain D39 genome suggesting that the differences in sRNA repertoires between these two serotypes may contribute to their strain-specific virulence properties. Finally, this study has identified 67 new sRNA candidates in strain D39, 28 out of which have been further validated, raising the total number of sRNAs that have been identified in strain D39 to 112.
2019-03-11 | GSE123437 | GEO
Project description:We were interested in determining if the LuxS signaling system contributed to the regulation of pneumococcal genes. To accomplish this, we compared the in vitro transcriptional profiles over time of S. pneumoniae D39, a serotype 2 strain of pneumococcus, with that of an isogenic deletion mutant, (delta)luxS, using a spotted S. pneumoniae- specific DNA microarray. For in vitro time course experiments, broth cultures of D39 and (delta)luxS were grown to low density (optical densities between ~0.01- 0.02), the cells were collected by centrifugation, washed 1x with sterile PBS, back diluted to ~3x105CFU/ ml into fresh broth, and immediately incubated at 37oC in a 5% CO2 chamber for 45 minutes. At this point, the first sample was collected. Subsequently, growth was monitored for approximately 8 generations by measuring both the optical density (OD600) and determining viable counts every 30 to 45 minutes. At these points samples were also harvested by centrifugation for RNA isolation and stored at -80oC until processing. For microarray probe synthesis, experimental samples were generated using 0.5ug total bacterial RNA isolated from either D39 or (delta)luxS strains at the various time points throughout the growth curve. These RNAs were used as templates for reverse transcription, and the resulting cDNAs were labelled with Cy5. The reference sample for each time course was generated from the D39 time zero RNA. Subsequent to reverse transcription, the resulting cDNAs were labelled with Cy3. The probes were mixed in hybridization buffer, heated to 99oC for at least 2 minutes, centrifuged briefly, and applied to the microarray. Hybridizations were carried out at 60oC for at least 24 hours.
2005-07-21 | E-SMDB-2173 | biostudies-arrayexpress
Project description:The stringent response is a conserved stress response employed by various bacteria to respond to and cope with conditions of amino acid starvation, carbon-source, fatty acid, oxygen/iron limitation, heat shock, antimicrobial challenge, and/or other environmental stressors. In S. aureus, the stringent response is mediated in part by enzyme RSH (relA spoT homologue) which synthesizes two nucleotide-based molecules, guanosine tetraphosphate (ppGpp) and pentaphosphate (pppGpp), collectively known as (p) ppGpp. This response is known to result in a number of diverse physiological and metabolic changes which we further characterized in the present study. Specifically, the global transcriptome of the RSH synthase mutant (rshsyn) was compared to the WT parent S. aureus strain (NCTC 8325) by RNA-Seq analysis under both normal and nutrient-limited media conditions. In order to mimic nutrient limitation, bacteria were treated with mupirocin, an antibiotic and isoleucine analogue known to lead to isoleucine starvation. In the WT strain, in response to mupirocin-induced isoleucine starvation, genes encoding protein synthesis machinery were suppressed while genes encoding amino acid biosynthetic machinery were upregulated. This response likely employed to compensate for amino acid deprivation, was not induced in the stringent response mutant. Furthermore, under starvation conditions, biofilm-related genes (i.e. icaA, icaC, fnbA, clfB, emp) were more upregulated in the WT than the stringent response mutant. Moreover, the effect of mupirocin caused a stronger upregulation of several proteases (i.e. sspA, sspP, sppB, aur), lipases (lip, geh) and phenol soluble modulin toxins in the WT as compared to the mutant. Overall, these results suggest that under starvation conditions, RSH plays an important role in helping the cell respond to nutritional stress as well as regulates the expression of numerous colonization and virulence factors.
2019-02-02 | GSE110170 | GEO
Project description:The bacterial second messenger c-di-AMP controls various cellular processes including potassium and osmolyte homeostasis. The c-di-AMP receptor protein DarB of Bacillus subtilis binds to the Rel protein and triggers the Rel-dependent stringent response. Here we report crystal structures of DarB in the ligand-free state and of DarB complexes with c-di-AMP, 3´3´-cGAMP and AMP. DarB consists of two CBS domains and forms a homo-dimer with a parallel, head-to-head assembly of the monomers. The DarB dimer binds two cyclic di-nucleotide molecules or two AMP molecules. Only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second one protrudes out of the donut-shaped protein. This enables DarB to bind also 3´3´-cGAMP, as only the adenine fits to the active site, but not the guanine. In absence of c-di-AMP DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes the interaction. The DarB crystal structures reveal no conformational changes upon c-di-AMP binding, hence, the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. A structural model of the DarB-Rel complex was erived from in silico docking, validated with a mass spectrometric analysis of the chemically cross-linked DarB-Rel complex and mutagenesis studies. Based on the predicted complex structure a mechanism of stringent response regulation by c-di-AMP is suggested.
2022-08-12 | PXD033346 | Pride
Project description:RelA/SpoT homologue (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of Streptococcus pneumoniae, Rel(Spn), can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 Deltarel(Spn) mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel(+)(Spn) and Deltarel(Spn) strains treated with mupirocin revealed rel(Spn)-independent (translation stress), rel(Spn)-dependent (stringent response) and Deltarel(Spn)-dependent changes, suggesting that rel(Spn) and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the rel(Spn)-dependent response included significant upregulation of the ply operon encoding pneumolysin toxin, whereas the Deltarel(Spn)-dependent response affected expression linked to the VicRK and CiaRH two-component systems. Finally, a D39 Deltarel(Spn) mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel(+)(Spn).
| S-EPMC2739083 | biostudies-literature
Project description:Rel is an enzyme involved in the metabolism of ppGpp. We investigated the role of ppGpp in Synechococcus elongatus PCC7942 in constant light and after exposure to darkness. We performed RNA-seq experiments using the rel- strain and wild type and show that ppGpp is required for regulation of global transcription in light and in darkness.
2017-12-12 | GSE103462 | GEO