Effects on small RNA MgrR in Escherichia coli
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ABSTRACT:
ORGANISM(S): Escherichia coli K-12 Escherichia coli
PROVIDER: GSE18935 | GEO | 2009/11/10
SECONDARY ACCESSION(S): PRJNA121049
REPOSITORIES: GEO
Similar Datasets
Project description:MgrR is a newly characterized Hfq dependent small RNA RNA. The expression of MgrR is regulated by Two component system, PhoPQ regulon, which senses low Mg2+ in environment. It has been reported that Hfq-binding sRNAs base pair with target RNAs, frequently leading to rapid degradation of target messages or, less frequently, to stabilization, both of which can be assayed by using microarrays. In order to search for the target genes of MgrR, we therefore examined the consequences of MgrR expression on mRNA abundance under two conditions. In condition 1, the chromosomal copy of mgrR was deleted and MgrR was expressed for 15â from an induced plac-mgrR plasmid and compared to cells carrying a vector induced for the same period. In condition 2, the expression of mRNAs was compared in wild-type cells (mgrR+) and the mgrR deletion strain, both grown in LB; because MgrR levels are fairly high under our normal growth conditions, this allowed analysis of both the direct and indirect (long-term) effects of MgrR. Experiment Overall Design: The strains used are either wild type which is derivatives of MG1655 or mgrR::kn knockout to restrict MgrR expression to the inducible pBR Plac-mgrR vector. The mgrR knockout mutants carried either pBR Plac-mgrR or the control vector pBR Plac. A Fresh colony of bacteria was inoculated in LB media with ampicilline at final concentration of 100ug/ml at 37°C with agitation. To induce MgrR expression, cultures carrying the pBR Plac-mgrR construct were grown to an O.D.600 of 0.5 and IPTG was added to the culture at a final concentration of 100uM. Both cultures for wild type and mgrR deletion mutant were grown to O.D. 600 of 0.5 and cells were collected. Total RNA was extracted from cells at the indicated time using the hot phenol procedure.
2009-11-14 | E-GEOD-18935 | biostudies-arrayexpress
Project description:In Neisseria meningitidis iron responsive gene regulation is mediated primarily by the Ferric Uptake Regulator (Fur) protein. When complexed with iron, Fur represses gene expression by preventing transcription initiation. Fur can also indirectly activate gene expression via the repression of regulatory small RNAs (sRNA). One such Fur-and iron-regulated sRNA, NrrF, was previously identified in N. meningitidis and shown to repress expression of the sdhA and sdhC genes encoding subunits of the succinate dehydrogenase complex. In the majority of Gram-negative bacteria sRNA-mediated regulation requires a cofactor RNA-binding protein (Hfq) for proper gene regulation and stabilization. In this study we examined the role of Hfq in NrrF-mediated regulation of the succinate dehydrogenase genes in N. meningitidis and the effect of an hfq- mutation on iron-responsive gene regulation more broadly. We first demonstrated that the stability of Nrrf as well as the regulation of sdhC and sdhA in vivo was unaltered in the hfq- mutant. Secondly, we established that iron responsive gene regulation of the Fur-regulated sodB gene was dependent on Hfq. Finally, we demonstrate that in N. meningitidis Hfq functions to control expression of both ORFs and intergenic regions via iron independent mechanisms. Collectively these studies demonstrate that in N. meningitidis iron and NrrF mediated regulation of sdhC and sdhA can occur independently of Hfq, although Hfq functions more globally to control regulation of other N. meningitidis genes primarily by iron-independent mechanisms. RNA was isolated from wild-type MC58 Neisseria meningitidis, from an hfq- mutant, and from a complemented hfq- mutant under both iron-replete and iron-deplete conditions. Three biological replicates were analyzed for each strain and condition were analyzed.
2010-05-14 | E-GEOD-20294 | biostudies-arrayexpress
Project description:The cross compatibility of functional PhoQ-PhoP mutants with altered specificity residues was analyzed by sort seq. We cloned and combinatorially combined 79 PhoQ* and 71 PhoP* variants, producing a library with a theoretical diversity of 5,609. The library was then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin (Fig. 2b). We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.
2019-07-27 | GSE120786 | GEO
Project description:cDNA microarray analysis to identify genes regulated by the RNA chaperone, Hfq. Four experiments were performed: 1/ Hfq+ vs Hfq- strains. 269 significantly differentially regulated genes were identified by SAM (Statistical Analysis of Microarrays), of which 120 changed more than 1.5 fold (48 increased and 72 decreased in hfq-). Amongst other genes, these experiments identified significant regulation of the sigma E and sigma 32 regulons. However, only genes induced by sigma E were similarly induced in hfq-; 8 operons repressed by sigma E were not repressed in hfq-. 2/ wt vs delta rseA. RseA is the antisigma factor for sigmaE. This comparison results in elevated steady-state levels of sigma E, and confirmed induction and repression of target regulon members. 3/ hfq+ vs hfq+ rpoE overexpression. RpoE encoding sigma E was overexpressed in an hfq+ background, confirming normal regulation of the sigma E regulon. 4/ hfq+ vs hfq- rpoE overexpression. Sigma E was overexpressed in an hfq- background. This demonstrated that 8 operons normally repressed by sigma E require hfq for this repression. The simple conclusion is that sigma E regulates small RNAs that, together with Hfq, bind target mRNAs and results in their rapid degradation. This study is detailed in Guisbert et al 2007 (J Bacteriol, 189:1963-73) Keywords: Genetic modification
2007-01-01 | GSE6444 | GEO
Project description:The specificity of the kinase-regulator interaction is driven by a limited set of interfacial residues in each protein that strongly. To identify combinations of these interface residues that are functional and potentially insulated from existing two-component signaling pathways in E. coli, we constructed a dual library of mutants in which the key, coevolving interface residues of a canonical two-component system, PhoQ and PhoP, were randomized. We used NNS codons to randomize six residues in PhoQ and five residues in PhoP, all of which lie at the interface formed by the two proteins in complex and that are critical to determining partner specificity in all two-component signaling pathways. To identify functional combinations of residues, we first grew the library of PhoQ-PhoP variants overnight in medium with low Mg2+, which activates PhoQ. Because cells must phosphorylate PhoP to grow when extracellular Mg2+ is limiting, this step enriches for functional PhoQ-PhoP variants. Variants that survived selection in limiting Mg2+ were then subjected to Sort-Seq, using fluorescence-activated cell sorting (FACS) and deep sequencing to quantify the signal responsiveness of variants in the library. To gauge the phosphorylation of PhoP in vivo, we used a fluorescent transcriptional reporter, PmgrB-yfp. In the presence of low extracellular Mg2+, functional PhoQ promotes the phosphorylation of PhoP and the production of YFP, whereas in the presence of high concentrations of Mg2+, PhoQ drives the dephosphorylation of PhoP, limiting the accumulation of YFP). The library was grown in each condition for 6 hours before sorting and sequencing. To identify variants that are signal responsive and drive YFP production specifically in low Mg2+, we sorted cells from each condition into 8 separate bins and deep sequenced the randomized regions of variants collected in each bin. We then calculated the frequency of each variant in each bin to yield the distributions of individual variants in low and high Mg2+, which were fit to Gaussians. From these fits, we assessed the mean level of YFP in each condition and the fold-induction, or signal responsiveness, of each variant detected in the library. The 11 codons / amino acids listed in this dataset refer to codons 12, 14, 15, 18, and 19 in PhoP and codons 284, 288, 289, 292, 302, and 303 in PhoQ, in that order.
2019-09-20 | GSE120780 | GEO
Project description:In response to low levels of magnesium (Mg2+), the PhoQP two component system induces the transcription of two convergent genes, one encoding a 31-amino acid protein denoted MgtS and the second encoding a small, regulatory RNA (sRNA) denoted MgrR. Previous studies showed that the MgtS protein interacts with and stabilizes the MgtA Mg2+ importer to increase intracellular Mg2+ levels, while the MgrR sRNA base pairs with the eptB mRNA thus affecting lipopolysaccharide modification. Surprisingly, we found overexpression of the MgtS protein also leads to induction of the PhoRB regulon. Studies to understand this activation showed that MgtS forms a complex with a second protein, PitA, a cation-phosphate symporter. Given that the additive effect of ∆mgtA and ∆mgtS mutations on intracellular Mg2+ concentrations seen previously is lost in the ∆pitA mutant, we suggest that MgtS binds to and blocks Mg2+ leakage through PitA under Mg2+-limiting conditions. Consistent with a detrimental role of PitA in low Mg2+, we also observe MgrR sRNA repression of PitA synthesis. Thus, PhoQP induces the expression of two convergent small genes in response to Mg2+ limitation whose products both repress PitA activity and levels to increase intracellular Mg2+ levels. Funded by NIH Intramural Program ZIA HD008855-11.
2018-10-03 | GSE120726 | GEO
Project description:Co-immunoprecipitation with endogenous Hfq3xFlag in exponential, stationary, non-induced and virulence induced conditions, followed by RNA-sequencing, revealed 1697 mRNAs and 208 ncRNAs associated with Hfq. We identified 56 new ncRNAs and validated 3 Hfq-dependent trans sRNAs on by Northern blot analysis. Interestingly, 55% of the ncRNAs were encoded antisense to a protein coding sequence. Abundance of 4 asRNAs and their corresponding target mRNAs was altered upon hfq, indicating a substantial influence of Hfq on asRNA-target interactions.
2014-12-04 | GSE59123 | GEO
Project description:<p>Aeromonas veronii (A. veronii) is a zoonotic pathogen. It causes clinically a variety of diseases such as dysentery, bacteremia, and meningitis. And it also brings huge losses to aquaculture. A. veronii has been documented as a multiple antibiotic resistant bacterium. Hfq (host factor for RNA bacteriophage Qβ replication) plays vital regulation roles in a variety of cell programs. The deletion of hfq in A. veronii showed an increased sensitivity to the trimethoprim, accompanying by the down regulation of the efflux pump related genes acrA and acrB in transcriptomic data. Coherently, the complementation of acrA and acrB in ∆hfq recovered the trimethoprim resistance. Besides, the productions of adenosine and guanosine were revealed to augment in ∆hfq in metabonomic data. Both wild type and ∆hfq conferred more sensitive to trimethoprim after appending 1 mM adenosine or guanosine to M9 medium. These results demonstrated that Hfq mediated trimethoprim resistance by elevating efflux pump expression and degrading adenosine, and guanosine metabolites. Collectively, Hfq is a potential target to tackle trimethoprim resistance in A. veronii infection.</p>
2020-02-19 | MTBLS1411 | MetaboLights
Project description:Bacterial small regulatory RNAs (sRNAs) regulate gene expression by base-pairing to their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, which binds sRNAs and mRNAs on different faces. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was identified as a novel silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and determined its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppresses all tested Hfq-dependent sRNA signaling. Chromosomal HqbA suppresses the signaling of the ChiX sRNA when the C-terminus of Hfq was deleted. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked interaction also interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, suggesting that HqbA is bifunctional, with separate roles for regulating via Hfq interaction and via acetylation of undefined substrates. Gel shift assays indicated that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs, but not high-affinity RNAs. Hfq-IP RNA-Seq in WT and chromosomal hqbA mutants led to the identification of two tRNA precursors, metZWV and proM, that were enriched in Hfq binding in the absence of HqbA interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.
2023-11-13 | GSE235194 | GEO
Project description:Transcriptional profiling of C. difficile 630E strain vs. an Hfq antisense after 7.5h of growth inTYt. Two-conditions experiments, 630E strain vs. Hfq antisense grown in TYt, 4 biological replicates for each condition.
2014-07-15 | E-GEOD-55273 | biostudies-arrayexpress