Transcription start site identification in wild type Escherichia coli
Ontology highlight
ABSTRACT: 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.
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Escherichia coli str. K-12 substr. MG1655
Project description:Experiment conducted to investigate the genetic factors associated with variable and heterogeneous non-planktonic in a mutant lacking rpoN unique to CFT073 bacteria. The aim was to try to understand why RpoN suppresses this heterogeneity in the wild-type during colony growth, but also asses why this phenotype does not occur in other pathogenic strains by also sequencing transcripts from EHEC EDL933. The rpoN mutant strains was complemented with plasmid-borne rpoN containing its native promoter to ensure any differences in gene expression could solely be attributed to the absence of rpoN alone.
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:Cappable-seq was used to map transcription start sites globally in wild type Bacillus subtilis. Total RNA was isolated from cells grown in LB media until exponential phase. RNA corresponding to transcription start sites was capped with a 5' biotin tag, which was used for enrichment via a pull down with streptavidin beads. Enriched RNA was converted to cDNA and then subjected to illumina sequencing.
Project description:In this study we generated 5'P libraries in wt and RNAse III mutant strains, grown to exponential and stationary phases. Libraries that retain short RNA fragments were also generated in both growth phases. After sequencing by Illumina NextSeq 500 system, reads were mapped to E. coli genome NC_000913.3. By comparing the read start counts per position in the wt and mutant strain libraries we identified the cleavage sites of RNase III.
Project description:As a comparison to tobramycin-treated P. aeruginosa biofilms, we investigated the response of planktonic P. aeruginosa to tobramycin by microarray. Experiment Overall Design: We included 2 control (untreated) cultures and 2 tobramycin-treated cultures. We used mid-exponential phase cultures of P. aeruginosa PA14. Replicate cultures were incubated in the presence or absence of 5 μg/mL tobramycin for 30 minutes at 37°C.
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:Investigation of comprehensive information about the expression level of RNA transcripts across the entire E.coli genome in mulitple growth conditions, including log-phase; stationary phase, heat shock and nitrogen-limiting condition. A fourteen chip study using total RNA recovered from four separate culture conditions of E.coli K12 MG1655. E.coli strains were harvested at mid-exponential phase with exception of stationary phase experiments. The high-density oligonucleotide tiling arrays used were consisted of 371,034 oligonucleotide probes spaced 25 bp apart (25-bp overlap between two probes) across the E. coli genome (NimbleGen). Experiments were conducted as three or more biological replicates (different cultures)
Project description:To study the regulatory outcome of Hfq-mediated sRNA-target interactions, we measured the change in gene expression following overexpression of each of five well-established sRNAs: GcvB, MicA, ArcZ, RyhB and CyaR. For each of the studied sRNAs we applied RNA-seq to two E. coli K-12 MG1655 strains: (1) a WT strain or a strain deleted of the sRNA gene; (2) a strain overexpressing the sRNA, either artificially from a plasmid or from the endogenous sRNA gene by changing the growth condition. For GcvB induction, the E. coli K-12 MG1655 Z1 gcvB::Cm, pZA12-gcvB and the E. coli K-12 MG1655 Z1 gcvB::Cm, pTP-011 strains were used. For MicA overexpression the E. coli K-12 MG1655lacIq pBRplac, pEF21-Hfq and the E. coli K-12 MG1655lacIq pMicA, pEF21-Hfq strains were used. For ArcZ induction the E. coli K-12 MG1655 Z1 arcZ::Cm, pZE12-ArcZ and the E. coli K-12 MG1655 Z1 arcZ::Cm, pJV300 strains were used. For RyhB overexpression the E. coli K-12 MG1655 ryhB::Cm and the E. coli K-12 MG1655 were used. For CyaR induction the E. coli K-12 MG1655 Z1 cyaR::Cm, pZE12-CyaR and the E. coli K-12 MG1655 Z1 cyaR::Cm, pJV300 strains were used. All E. coli strains used in this study were grown overnight in Luria Bertani (LB) medium at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB medium, and re-grown with shaking at 37 °C to exponential phase, for GcvB (OD600 = 0.3) and for RyhB (OD600 = 0.5), or to stationary phase, for ArcZ WT, ArcZ M1, ArcZ M2 and CyaR (OD600 = 1.0) and for MicA (grown for 6 hr). For induction of ArcZ and GcvB, IPTG was added (1mM, 20 min). MicA was constitutively expressed and further induced at the end of growth with IPTG (1mM, 20 min). For induction of RyhB, the iron chelator 2,2'-Dipyridyl was added (200 μM, 30 min).
Project description:DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM's DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential ÏE cell envelope stress pathway is dispensable in ÎvchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some cell envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex cell circuits to control critical housekeeping processes. Duplicates were used for all samples. For each strain background (C6706 and O395), there were control (Wildtype) samples and experimental samples (VchM knockout)