Effect of hfq-deletion on Salmonella transcriptome
Ontology highlight
ABSTRACT:
ORGANISM(S): Salmonella enterica
SUBMITTER: Sacha Lucchini
PROVIDER: E-GEOD-8985 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial âcoreâ small RNA of unknown function. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of >16% (â¥750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs suggesting that the unprecedented degree of pleiotropic regulation might in part be caused by titration of Hfq binding by ArcZ. This study used three different approaches to identify target genes and biological role of the small RNA ArcZ in Salmonella Typhimurium. Transcriptomic analysis of ArcZ overexpression: Strain JVS-0082 (ÎarcZ) was transformed with plasmids pJV300 (control) and pKP48-1 (parcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptomic analysis of arcZ mutant strain: Strains JVS-007 (WT) and JVS-0082 (ÎarcZ) were transformed with plasmid pJV300 (control) and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptional effects of ArcZ pulse expression: Strain SL1344 was transformed with plasmids pKP8-35 (pBAD-control) and pKP4-13 (pBAD-ArcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture to an OD600 of 1.5. Expression of the insert was induced with L-arabinose (0.2% final concentrations) for 10 min. Three biological replicates were performed for each strain/condition.
2009-10-19 | E-GEOD-17771 | biostudies-arrayexpress
Project description:It is well established that small noncoding RNAs (sRNAs) of bacteria recognize the 5â?? regions of target mRNAs, generally at the ribosome binding site. Until now, the translated coding sequence (CDS) of mRNA appeared to be refractory to sRNA interactions. We have discovered that Salmonella MicC RNA silences ompD mRNA by targeting the CDS, at codons 23-26. Analyses of MicC-ompD RNA complexes in vitro, and of chimeric sRNAs and ompD reporter fusions in vivo, show that interactions are confined to the CDS, and that a â?¤12 bp RNA duplex involving the conserved 5â?? end of MicC is essential and sufficient for target repression. MicC cannot act as a translational repressor at this downstream position being unable to inhibit 30S or 70S ribosome activity on the ompD mRNA. Instead, MicC accelerates RNase E-dependent ompD mRNA decay. The degradosome contributes to target destabilization, and facilitates the efficient degradation of processed ompD mRNA. Our data show that bacterial gene silencing by sRNAs can take place downstream of the translational initiation site by triggering irreversible mRNA decay. This ability of sRNAs allows targets in the CDS to be silenced without interference from the strong RNA helicase activity of elongating ribosomes. To determine the targets of the small regulatory RNA MicC in S. Typhimurium, we looked at the effect of a short pulse of MicC over-expression on the Salmonella transcriptome. To achieve over-expression, the micC gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control. 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674â??1688.
2010-05-16 | E-GEOD-16540 | biostudies-arrayexpress
Project description:StpA is a paralogue of the nucleoid associated protein H-NS that is conserved in a range of enteric bacteria and had no known function in Salmonella enterica serovar Typhimurium. Here, we show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA-dependent genes of S. Typhimurium are a specific subset of the H-NS regulon that are predominantly under the positive control of sigma38 (RpoS), CRP-cAMP and PhoP. The regulatory role of StpA varied at different growth phases; StpA only controlled sigma38 levels at mid-exponential phase when it prevented inappropriate activation of sigma38 during rapid bacterial growth. In contrast, StpA only activated the CRP-cAMP regulon during late exponential phase. The effect of stpA deletion on S. Typhimurium gene expression during growth in LB was analysed at 4 different time points (early-log, mid-log, late-log, and stationary phase) where the gene expression profile of the stpA-deletion strain was compared to that of the parental strain. Between two and three biological replicates were performed for each strain and time point. For this study, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality.
2009-10-27 | E-GEOD-18424 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE18424: The effect of stpA deletion on S. Typhimurium gene expression during growth in rich medium GSE18428: StpA prevents RpoS-dependent transcription during mid-exponential growth in S. Typhimurium GSE18450: Identification of StpA-binding sites on the Salmonella genome Refer to individual Series
2010-06-21 | E-GEOD-18452 | biostudies-arrayexpress
Project description:We used high-throughput methods to discover the Salmonella RNAs that are targeted by the bacterial Sm-like protein, Hfq. Our generic approach, using chromosomal epitope tagging and coIP-on-chip will also be useful to identify the post-transcriptional regulons of many other RNA-binding proteins. Keywords: coIP-chip To identify the direct Hfq RNA targets, we co-immunoprecipitated RNA from a strain expressing a 3xFLAG-tagged Hfq-protein using an anti-3xFLAG antibody. As a negative control, we used the same antibody (anti-3xFLAG) to co-immunoprecipitate RNA from an isogenic strain expressing the non-tagged Hfq-protein. Two independent biological replicates were generated.
2010-06-20 | E-GEOD-10149 | biostudies-arrayexpress
Project description:We used ChIP-chip analysis to identify where the nucleoid associated protein StpA binds on the Salmonella Typhimurium genome. We identified 285 chromosomal regions bound by StpA, ranging in size from 400 to 3500 base pairs and containing 572 genes. Comparison of the ChIP data revealed that StpA displayed an identical binding profile to H-NS; all genomic regions associated with StpA were also bound by H-NS. Of the 88 StpA-dependent genes that are de-repressed in the absence of StpA, only 25 were bound by StpA, suggesting that StpA generally regulates gene expression indirectly. To identify the direct StpA targets, we co-immunoprecipitated DNA from a strain expressing a 3xFLAG-tagged StpA-protein using an anti-3xFLAG antibody. As a negative control, we used the same antibody (anti-3xFLAG) to co-immunoprecipitate DNA from an isogenic strain expressing the non-tagged StpA-protein. Three independent biological replicates were generated. For comparative purposes we also performed the co-immunoprecipitation on wild type Salmonella using a monoclonal anti-H-NS antibody. In that case, the negative control was obtained by performing the co-immunoprecipitation protocol on wild type Salmonella without adding antibody.
2009-10-28 | E-GEOD-18450 | biostudies-arrayexpress
Project description:S. Typhimurium 112910a parental strain and an isogenic M-NM-^TscsA strain were grown to stationary phase in SPI2 inducing minimal medium (MM5.8 - Erikkson et al., 2006 Inf & Imm., 74:1243) with or without supplementation with 1M-BM-5M cortisol 4 samples (stat phase wt, stat phase wt + 1M-BM-5M cortisol, stat phase M-NM-^TscsA, stat phase M-NM-^TscsA + 1M-BM-5M cortisol) and 3 biological replicates for each sample.
2014-02-28 | E-GEOD-55430 | biostudies-arrayexpress
Project description:The expression profile of an S. Typhimurium mutant strain unable to synthesise ppGpp (relAspoT deletions) was compared to the wild-type strain. The effect of ppGpp on virulence gene expression was studied under 4 different growth conditions that induce virulence gene expression. Keywords: genetic modification Study comprised 4 separate experiments (with 2 strains and 4 replicates for each strain per growth condition). The experiments were indirect comparisons using Salmonella genomic DNA as the comparator which also acted as the control for spot quality.
2010-06-11 | E-GEOD-4631 | biostudies-arrayexpress
Project description:Transcriptional profiling of Salmonella Typhimurium SL1344 parental starin and isogenic M-bM-^HM-^FrelA, M-bM-^HM-^FspoT ppGpp null strain grown in LB medium with RNA samples talken at AD600=1.0 (mid log, ML), 2.3 (early stationary phase, ESP), 3.0 (mid stationary phase MSP) and 3.6 (Late stationary phase (LSP) Each array used labelled cDNA against a common genomic DNA reference. Triplicate biologically independent RNA samples were arrayed for each of the 2 strains at each of the 4 growth phases
2013-07-03 | E-GEOD-48478 | biostudies-arrayexpress
Project description:SgrS RNA is a model for the large class of Hfq-associated small RNAs that act to post-transcriptionally regulate bacterial mRNAs. The function of SgrS is well-characterized in non-pathogenic Escherichia coli where it was originally shown to counteract glucose-phosphate stress by acting as a repressor of ptsG mRNA which encodes the major glucose transporter. We have discovered new SgrS targets in Salmonella Typhimurium, a pathogen related to E. coli, which recently acquired a quarter of all genes by horizontal gene transfer. We demonstrate that the conserved short seed region of SgrS that recognizes ptsG was recruited to target the Salmonella-specific sopD mRNA of a secreted virulence protein. The SgrS-sopD interaction is exceptionally selective; we find that sopD2 mRNA whose gene arose from sopD duplication during Salmonella evolution, is deaf to SgrS owing to a non-productive G:U pair in the potential SgrS-sopD2 RNA duplex versus G:C in SgrS-sopD. In other words, SgrS discriminates the two virulence factor mRNAs at the level of a single hydrogen bond. Our study suggests that bacterial pathogens employ their large suites of conserved Hfq-associated regulators to integrate horizontally acquired genes into existing post-transcriptional networks, just as conserved transcription factors are recruited to tame foreign genes at the DNA level. The results graphically illustrate the importance of the seed regions of bacterial small RNAs to select new targets with high fidelity, and argue that target predictions must consider all-or-none decisions by individual seed nucleotides. To determine the targets of the small regulatory RNA SgrS in S. Typhimurium, we looked at the effect of a short pulse of SgrS over-expression on the Salmonella transcriptome. To achieve over-expression, the sgrS gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control. 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674–1688.
2012-01-20 | E-GEOD-34851 | biostudies-arrayexpress