RNA-seq analysis of MG1655 Escherichia coli (Wild-type and mutant lacking the RNA chaperone Hfq) experiencing long-term nitrogen starvation in Gutnick minimal media
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ABSTRACT: RNA-seq was carried out to compare the transcriptomes of wild-type MG1655 E coli with mutant lacking the prominent RNA chaperone, Hfq, in bacteria that had been exposed to short-term (20min, N-) and long-term (24hr, N-24) nitrogen starvation, and following replenishment of nitrogen to long-term starved bacteria (~2hrs, N-24+2) in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of Hfq was to bacteria experiencing nitrogen starvation.
Project description:RNA-seq was carried out to compare the transcriptomes of wild-type BW25113 E coli with a mutant lacking the ubiquitous RNA chaperone Hfq, in bacteria that had been exposed to long-term (24hr) nitrogen starvation in Gutnick minimal media. The mutation was also complemented with wild-type Hfq or point mutants of Hfq (Q8A, V22A & G34A), provided by the arabinose inducible pBAD plasmid. The aim of this was to understand the effect of different point mutations of Hfq on the transcriptome, and specifically sRNA stability under physiological conditions.
Project description:RNA-seq was carried out to compare the transcriptomes of wild-type MG1655 E coli with mutant lacking the prominent sRNA, SdsR, in bacteria that had been exposed to long-term (24hr) nitrogen starvation in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of SdsR was to bacteria experiencing long-term nitrogen starvation.
Project description:RNA interaction by ligation and sequencing (RIL-seq) was carried out to compare the Hfq-RNA-RNA interactome of MG1655 E coli bacteria that grown in Gutnick minimal media. Wild-type and hfq::3xFLAG tagged K12 E.coli were grown in Gutnick minimal media with 3mM NH4Cl as the sole nitrogen source, and cell samples were taken during exponential growth (N+), ~20min following N-runout and induction of growth arrest (N-), 24hr following N-runout and induction of growth arrest (N-24) and 2hr following addition of NH4Cl to N-24 cells (N-24+2). All sampling was performed in triplicate for each strain and condition
Project description:RNA-seq was carried out to compare the transcriptomes of wild-type BW25133 E coli with a mutant lacking the conserved serine/threonine kinase, YeaG, with the aim of understanding how YeaG contributes to the transcriptional response to sustained nitrogen starvation, and identify pathways affected by YeaG. We also compare the wild-type transcriptome during nitrogen-replete, following nitrogen starvation, and under 24 h of sustained nitrogen starvation to identify pathways involved in the response to nitrogen run out, and also in adaptation to sustained nitrogen starvation.
Project description:RNA-seq data to compare the transcriptomes of wild-type and hfq mutant E. coli strain MG1655 experiencing nitrogen starvation for 20 min (N-) and 24 hours (N-24). To look at the RNA expression profile of E. coli during Nitrogen starvation and comparing how this changes in bacteria lacking Hfq at two specific timepoints; short-term Nitrogen starvation, N- (20min into starvation), and long-term Nitrogen starvation, N-24 (24hours into starvation).
Project description:UV-crosslinking and high througput sequencing of cDNAs (CRAC) was used to map the binding sites Hfq in enterohaemorhaggic E. coli (EHEC). Hfq was tagged with a His-FLAG dual affintiy tag and UV crosslinked after growth in the MEM-HEPES media essentailly as per Granneman et al (2009) PNAS. We additionally crosslinked Hfq in non-pathogenic E. coli K12 str. MG1655 grown in LE media. Hfq-RNA complexes were purified and trimmed using RNase A/T1. RNA fragments were isolated and converted to cDNA, PCR amplified and sequenced using Illumina Solexa GAxII and HiSeq2000 platforms. His-FLAG tagged Hfq and untagged controls were cultured to an OD of 0.8 and crosslinked with UV-C. Five replicates of tagged EHEC Hfq and 2 replicates of untagged Hfq were crosslinked. We have additionally crosslinked 2 replicates each of E. coli K12 tagged and untagged Hfq.
Project description:RNAseq was used to map transcription start sites globally in wild type Escherichia coli. Total RNA was isolated from cells grown in LB media until exponential phase.
Project description:Proteomics was carried out to compare the proteomes of wild-type MG1655 E coli with mutants lacking either prominent sRNA, SdsR, OxyS or ZbiJ, or lacking the ubiquitous RNA chaperone proteins hfq, in bacteria that had been exposure to long-term (24hr) nitrogen starvation in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of these three sRNA was to bacteria experiencing long-term nitrogen starvation.
Project description:The RNA-binding protein Hfq is a global regulator, which controls diverse cellular processes in bacteria. To begin understanding the role of Hfq in the Sinorhizobium meliloti-Medicago truncatula nitrogen-fixing symbiosis, we defined free-living and symbiotic phenotypes of an hfq mutant. Over 500 transcripts were differentially accumulated in the hfq mutant of S. meliloti Rm1021 when grown in a shaking culture.
Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.