GSM4543918: BL21_Fur_Fe_1; Escherichia coli; OTHER - SRA

SRX8320974: GSM4543918: BL21_Fur_Fe_1; Escherichia coli; OTHER
1 ILLUMINA (Illumina HiSeq 4000) run: 3.6M spots, 111M bases, 41.3Mb downloads

Submitted by: NCBI (GEO)

Study: The Fur Pan-regulon in E. coli shows diversity in strain-specific sets of target genes

PRJNA631626 • SRP261075 • All experiments • All runs

show Abstracthide Abstract

Regulons for many transcription factors have been elucidated in model strains leading to an understanding of their role in producing physiological states. Comparative analysis of a regulon and its target genes between different strains of the same species is lacking. Ferric uptake regulator (Fur), involved in iron homeostasis, is one of the most conserved TFs, and is present in a wide range of bacteria. Using ChIP-exo experiments, we performed a comprehensive study of Fur binding sites in nine Escherichia coli strains with different lifestyles. 79 of the 431 target genes (18%) found belong to Fur's core regulon, comprising genes involved in ion transport and metabolism, energy production and conversion, and amino acid metabolism and transport. 179 of the target genes (42%) comprise the accessory regulon, most of which were related to cell wall structure and biogenesis, and virulence factor pathways. The remaining target genes (173 or 40%) were in the unique regulon, with gene functions that were largely unknown. Furthermore, deletion of the fur gene led to distinct phenotypes in growth, motility, antibiotic resistance, and siderophore production. These results provide a more complete understanding of how Fur regulates a set of target genes with surprising variation in closely related bacteria. Overall design: Identification of genome-wide bindings for nine different E. coli strains using ChIP-exo technology

Sample: BL21_Fur_Fe_1

SAMN14884966 • SRS6640289 • All experiments • All runs

Organism: Escherichia coli BL21(DE3)

Library:

Instrument: Illumina HiSeq 4000

Strategy: OTHER

Source: GENOMIC

Selection: other

Layout: SINGLE

Construction protocol: Lysates were clarified from sonicated uncharacterized transcripton factors-DNA complexes were isolated with myc antibody. ChIP-exo experiment was performed following the procedures: to identify each TF candidate binding maps in vivo, we isolated the DNA bound to each TF candidate from formaldehyde cross-linked E. coli cells by chromatin immunoprecipitation (ChIP) with the specific antibodies that specifically recognizes myc tag (9E10, Santa Cruz Biotechnology), and Dynabeads Pan Mouse IgG magnetic beads (Invitrogen) followed by stringent washings as described previously [42]. ChIP materials (chromatin-beads) were used to perform on-bead enzymatic reactions of the ChIP-exo method [11]. Briefly, the sheared DNA of chromatin-beads was repaired by the NEBNext End Repair Module (New England Biolabs) followed by the addition of a single dA overhang and ligation of the first adaptor (5'-phosphorylated) using dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs), respectively. Nick repair was performed by using PreCR Repair Mix (New England Biolabs). Lambda exonuclease- and RecJf exonuclease-treated chromatin was eluted from the beads and the protein-DNA cross-link was reversed by overnight incubation at 65oC. RNAs- and Proteins-removed DNA samples were used to perform primer extension and second adaptor ligation with following modifications. The DNA samples incubated for primer extension as described previously [12] were treated with dA-Tailing Module (New England Biolabs) and NEBNext Quick Ligation Module (New England Biolabs) for second adaptor ligation. The DNA sample purified by GeneRead Size Selection Kit (Qiagen) was enriched by polymerase chain reaction (PCR) using Phusion High-Fidelity DNA Polymerase (New England Biolabs). The amplified DNA samples were purified again by GeneRead Size Selection Kit (Qiagen) and quantified using Qubit dsDNA HS Assay Kit (Life Technologies). Quality of the DNA sample was checked by running Agilent High Sensitivity DNA Kit using Agilent 2100 Bioanalyzer (Agilent) before sequenced using HiSeq (Illumina) in accordance with the manufacturer's instructions. Each modified step was also performed in accordance with the manufacturer's instructions. ChIP-exo experiments were performed in biological duplicate.

Experiment attributes:

GEO Accession: GSM4543918

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 3.6M spots, 111M bases, 41.3Mb

Run	# of Spots	# of Bases	Size	Published
SRR11767681	3,579,240	111M	41.3Mb	2023-08-05

ID:: 10824972

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