Project description:Chaperones have essential role in assist nascent peptides folding, prevent proteins aggregation and maintain cellular protein homeostasis. Considering spatial and temporal features of chaperones regulating in vivo, changes in single or combined chaperone-depleted E.coli strain is needed to be put into understand at transcriptional level. Here, we utilized expression microarrays to investigate global transcriptional response upon deletion of single or multiple chaperones in E. coli for understanding the transcriptional network affected by chaperones. To identify prokaryotic expression profiles in deletion of chaperones, several E.coli mutants were constructed in the following: Z116 (△tig 37℃), Z125(△dnaK37℃), Z625(△tig△dnaK 37℃ or 30℃), Z629(△tig△dnaK 30℃), NM (C-domain of tig was deleted 37℃), MC (N-domain of tig was deleted 37℃), NC (M-domain of tig was deleted 37℃). Two types of cDNA mixtures containing Cy3-labeled (or Cy5-labeled) control DNA (from BW25113) and Cy5-labeled (or Cy3-labeled) DNA targets (from mutant strain) were hybridized with E.coli microarrays in a dye-swap strategy. E.coli gene expression data of chaperone DnaK deletant compared control WT (BW25113). Please note that other mutant microarray data will be added in the future.
Project description:Chaperones have essential role in assist nascent peptides folding, prevent proteins aggregation and maintain cellular protein homeostasis. Considering spatial and temporal features of chaperones regulating in vivo, changes in single or combined chaperone-depleted E.coli strain is needed to be put into understand at transcriptional level. Here, we utilized expression microarrays to investigate global transcriptional response upon deletion of single or multiple chaperones in E. coli for understanding the transcriptional network affected by chaperones. To identify prokaryotic expression profiles in deletion of chaperones, several E.coli mutants were constructed in the following: Z116 (△tig 37℃), Z125(△dnaK37℃), Z625(△tig△dnaK 37℃ or 30℃), Z629(△tig△dnaK 30℃), NM (C-domain of tig was deleted 37℃), MC (N-domain of tig was deleted 37℃), NC (M-domain of tig was deleted 37℃). Two types of cDNA mixtures containing Cy3-labeled (or Cy5-labeled) control DNA (from BW25113) and Cy5-labeled (or Cy3-labeled) DNA targets (from mutant strain) were hybridized with E.coli microarrays in a dye-swap strategy.
Project description:YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet undefined function in the oxidative stress response. YajL protects cells against oxidative stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. As compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA-repair proteins, RNA chaperones and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism.
Project description:YajL is the most closely related Escherichia coli homolog of Parkinsonism-associated protein DJ-1, a protein with a yet undefined function in the oxidative stress response. YajL protects cells against oxidative stress-induced protein aggregation and functions as a covalent chaperone for the thiol proteome, including FeS proteins. To clarify the cellular responses to YajL deficiency, transcriptional profiling of the yajL mutant was performed. As compared to the parental strain, the yajL mutant overexpressed genes coding for chaperones, proteases, chemical chaperone transporters, superoxide dismutases, catalases, peroxidases, components of thioredoxin and glutaredoxin systems, iron transporters, ferritins and FeS cluster biogenesis enzymes, DNA-repair proteins, RNA chaperones and small regulatory RNAs. It also overexpressed the RNA polymerase stress sigma factors sigma S (multiple stresses) and sigma 32 (protein stress) and activated the OxyR and SoxRS oxidative stress transcriptional regulators, which together trigger the global stress response. The yajL mutant also overexpressed genes involved in septation and adopted a shorter and rounder shape characteristic of stressed bacteria. Biochemical experiments showed that this upregulation of many stress genes resulted in increased expression of stress proteins and improved biochemical function. Thus, protein defects resulting from the yajL mutation trigger the onset of a robust and global stress response in a prokaryotic model of DJ-1-associated Parkinsonism. We performed microarray analysis of the transcriptome response of the yajL mutant and its parental strain in the exponential phase of growth (grown in aerobiosis in LB medium to OD600 = 0.3) in the absence of any exogenous stress. Two replicates per strain (wild type, yajL mutant).
Project description:Profile data for > 34'000 analyses of about 3800 E. coli single gene deletion mutants from the KEIO collection by flow injection - time-of-flight analysis in positive and negative mode. Each injection was recalibrated and flattened to a single profile. Profiles are stored as [m/z intensities] pairs in plain text. Files are described in the excel workbooks.
Project description:B-methylthiolation of the Escherichia coli Ribosomal Protein S12 Regulates Anaerobic Gene Expression. B-methylthiolation is a unique post-translational modification (PTM) that maps to a conserved Asp 88 of the bacterial ribosomal protein S12. This modification is phylogenetically conserved in several bacteria yet has not been identified on other proteins. We use microarrays to delineate the association of prokaryotic ribosomal protein PTM to the regulation of genes.
Project description:Elucidating genome-scale regulatory networks requires a comprehensive collection of gene expression profiles, yet measuring gene expression responses for every transcription factor (TF)-gene pair in living prokaryotic cells remains challenging. Here, we develop pooled promoter responses to TF perturbation sequencing (PPTP-seq) via CRISPR interference to address this challenge. Using PPTP-seq, we systematically measure the activity of 1372 Escherichia coli promoters under single knockdown of 183 TF genes, illustrating more than 200,000 possible TF-gene responses in one experiment. We perform PPTP-seq for E. coli growing under three different media. The PPTP-seq data reveal robust steady-state promoter activities under most single TF knockdown conditions. PPTP-seq also enables identifications of new TF auto-regulatory responses and complex transcriptional control on one-carbon metabolism. We further find context-dependent promoter regulation by multiple TFs whose relative binding strengths determined promoter activities. Additionally, PPTP-seq reveals different promoter responses under different growth media, suggesting condition-specific gene regulation. Overall, PPTP-seq provides a powerful method to examine genome-wide transcriptional regulatory networks and can be potentially expanded to reveal gene expression responses to other genetic elements.