biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsEwelina BoguszewskaIllumina NovaSeq 6000ChIP-seqEscherichia coli str. K-12 substr. MG1655Escherichia coli str. K-12 substr. MG1655https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14964The ubiquitous protein DnaA is a chromosomal DNA replication initiator and transcription factor. It binds to 9-bp DNA sites called \"DnaA-boxes\" that are localized within the replication origin (oriC) and throughout chromosomal DNA, usually in the proximity of the promoter regions. During stress, bacteria undergo metabolic shifts that halt their growth and activate survival mechanisms. One outcome of these shifts is the accumulation of long chains of polyphosphate (polyP), an evolutionarily conserved linear polymer composed of up to 1,000 phosphate groups. It was previously identified that DnaA and the Lon protease bind to polyP, which stimulates Lon for the polyP-dependent DnaA proteolysis. We assumed that it have an impact not only on DnaA intracellular concentration but might also affect DNA binding pattern. To investigate this hypothesis and to analyze the DNA interaction profile of DnaA molecules remaining in stressed cells, we performed Chromatin Immunoprecipitation coupled with DNA sequencing (ChIP-seq).biostudies-arrayexpressSample Collection - Amino acid starvation was induced using serine hydroxamate (SHX) treatment. E. coli cells were grown in LB medium at 30°C with aeration until the optical density at 600 nm (OD600) reached 0.6 (before stress sample). SHX was added at a final concentration of 4 mM, and the cultures were incubated for an additional 2 h (stress sample). Cells were harvested by centrifugation, washed twice in LB medium to remove residual SHX, and resuspended in fresh LB medium. Recovery was monitored by growing the cells for 30 in LB medium (recovery sample). 40 ml of bacterial cultures (before stress, during stress or recovery) were treated with 1% formaldehyde for 20 minutes at room temperature. Crosslinking was stopped by adding glycine at final concentration 500 mM and incubating for 5 minutes. The cultures were then centrifuged and washed with TBS.Nucleic Acid Extraction - Following crosslinking, cells were lysed by lysozyme treatment and sonication. A 2% aliquot of each lysate was used as the input DNA controls (input samples). Immunoprecipitation was performed using anti-DnaA rabbit antibodies and Protein A Sepharose beads pre-blocked with a blocking buffer (0.2% BSA, 1 mg/mL PVP, 1×TBS). The immunoprecipitated DNA and input DNA samples were decrosslinked and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). Purified DNA was subsequently analyzed using Next-Generation Sequencing (ChIP-seq). ChIP-seq were performed in three biological replicates for each condition.Sequencing - DNA sequencing of ChIP and input samples was performed by Novogene (United Kingdom) using the Illumina NovaSeq 6000 ChIP-seq protocol, which included sample quality control, library preparation and quality control, sequencing 150 bp from both ends of amplified fragments (NovaSeq PE150; 150 bp paired-end with directional protocol; 6G raw data per sample), ended with reads quality control.Library Construction - Library preparation was performed by Novogene (United Kingdom)OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesEwelina BoguszewskafalseChIP-seq experiment: DnaA binding to E. coli chromosome before stress, during stress, and stress recoveryThe ubiquitous protein DnaA is a chromosomal DNA replication initiator and transcription factor. It binds to 9-bp DNA sites called \"DnaA-boxes\" that are localized within the replication origin (oriC) and throughout chromosomal DNA, usually in the proximity of the promoter regions. During stress, bacteria undergo metabolic shifts that halt their growth and activate survival mechanisms. One outcome of these shifts is the accumulation of long chains of polyphosphate (polyP), an evolutionarily conserved linear polymer composed of up to 1,000 phosphate groups. It was previously identified that DnaA and the Lon protease bind to polyP, which stimulates Lon for the polyP-dependent DnaA proteolysis. We assumed that it have an impact not only on DnaA intracellular concentration but might also affect DNA binding pattern. To investigate this hypothesis and to analyze the DNA interaction profile of DnaA molecules remaining in stressed cells, we performed Chromatin Immunoprecipitation coupled with DNA sequencing (ChIP-seq).2026-03-13T00:00:00Z2026-03-13T02:02:31.401Z2025-03-20T14:48:01.347ZE-MTAB-14964ERP170618EFO_0002944EFO_0004170EFO_0002692EFO_0005518EFO_0004184