Project description:The ability to respond to stress is at the core of an organismâs survival. The hormones epinephrine and norepinephrine play a central role in stress responses in mammals, which require the synchronized interaction of the whole neuroendocrine system. Bacteria also sense and respond to epinephrine and norepinephrine as a means to gauge the metabolic and immune state of the host. Mammalian adrenergic receptors are G-coupled protein receptors (GPCRs), bacteria, however, sense these hormones through histidine sensor kinases (HKs). HKs autophosphorylate in response to multiple signals and transfer this phosphate to response regulators (RRs). Two bacterial adrenergic receptors have been identified in EHEC, QseC and QseE, with QseE being downstream of QseC in this signaling cascade. We mapped the QseC signaling cascade in the deadly pathogen enterohemorrhagic E. coli (EHEC), which exploits this signaling system to promote disease. Through QseC, EHEC activates expression of metabolic, virulence and stress response genes, synchronizing the cell response to these stress hormones. Coordination of these responses is achieved by QseC phosphorylating three of the thirty two EHEC RRs. The QseB RR, which is QseCâs cognate RR, activates the flagella regulon which controls bacteria motility and chemotaxis. The QseF RR, which is phosphorylated by the QseE adrenergic sensor, coordinates expression of virulence genes involved in formation of lesions in the intestinal epithelia by EHEC, and the bacterial SOS stress response. The third RR, KdpE, controls potassium uptake, osmolarity response, and also the formation of lesions in the intestine. Adrenergic regulation of bacterial gene expression shares several parallels with mammalian adrenergic signaling having profound effects in the whole organism. Understanding adrenergic regulation of a bacterial cell is a powerful approach to study the underlying mechanisms of stress and cellular survival. Experiment Overall Design: Microarray comparisons reveal some trends with respect to signaling cascades. Comparative methods were used to identify networks.
Project description:Analysis of effect of deletion of the two adrenergic kinases QseC and QseE on gene expression of EHEC 8624 in the absence and presence of epinephrine
Project description:We replaced the natural pnp locus with the human cDNA and studied the transcriptomes of 3 strains, namely the wt pnp+ (C-1a), the mutant with pnp ORF deletion (C-5691) and the strain with the substitution of the bacterial ORF with the human one (C-6001).
Project description:Bacterial transcription factors (TFs) regulate gene expression to adapt to changing environments; when combined, the TF’s regulatory actions comprise transcriptional regulatory networks (TRNs). The chromatin immunoprecipitation (ChIP) assay is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It enables the genome-wide study of transcription factor binding sites (TFBSs) and gene regulation. Here, we present the genome-wide binding for major TFs in E. coli K-12 MG1655.
Project description:These E. coli strains were grown with various signaling molecules and the expression profiles were determined. Keywords: addition of quorum and host hormone signals
Project description:Using a high-density tiling array containing 25-mer oligonucleotides at the resolution of one every four base pairs across the entire genome, we developed In vivo Protein Occupancy Display (IPOD), a technology that reveals protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites. Application to Escherichia coli reveals thousands of protein occupancy peaks, highly enriched within and in close proximity to non-coding regulatory regions.
Project description:Analysis of effect of deletion of the two adrenergic kinases QseC and QseE on gene expression of EHEC 8624 in the absence and presence of epinephrine A double mutant of qseC and qseE in the EHEC 8624 was grown in the absence or presence of epinephrine to OD600=1.0 in DMEM then processed according to manufacturer's specifications: http//www.affymetrix.com/support/technical/manual/expression_manual.affx
Project description:Bacterial stress responses have been studied at the phenotypic, transcriptional, and translational levels, demonstrating the presence of an “alarm” phase immediately after stress exposure. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic RNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the alarm phase of stress adaptation.
Project description:The ability to respond to stress is at the core of an organism’s survival. The hormones epinephrine and norepinephrine play a central role in stress responses in mammals, which require the synchronized interaction of the whole neuroendocrine system. Bacteria also sense and respond to epinephrine and norepinephrine as a means to gauge the metabolic and immune state of the host. Mammalian adrenergic receptors are G-coupled protein receptors (GPCRs), bacteria, however, sense these hormones through histidine sensor kinases (HKs). HKs autophosphorylate in response to multiple signals and transfer this phosphate to response regulators (RRs). Two bacterial adrenergic receptors have been identified in EHEC, QseC and QseE, with QseE being downstream of QseC in this signaling cascade. We mapped the QseC signaling cascade in the deadly pathogen enterohemorrhagic E. coli (EHEC), which exploits this signaling system to promote disease. Through QseC, EHEC activates expression of metabolic, virulence and stress response genes, synchronizing the cell response to these stress hormones. Coordination of these responses is achieved by QseC phosphorylating three of the thirty two EHEC RRs. The QseB RR, which is QseC’s cognate RR, activates the flagella regulon which controls bacteria motility and chemotaxis. The QseF RR, which is phosphorylated by the QseE adrenergic sensor, coordinates expression of virulence genes involved in formation of lesions in the intestinal epithelia by EHEC, and the bacterial SOS stress response. The third RR, KdpE, controls potassium uptake, osmolarity response, and also the formation of lesions in the intestine. Adrenergic regulation of bacterial gene expression shares several parallels with mammalian adrenergic signaling having profound effects in the whole organism. Understanding adrenergic regulation of a bacterial cell is a powerful approach to study the underlying mechanisms of stress and cellular survival.
Project description:YbjN, an enterobacteria-specific protein, is a multicopy suppressor of ts9 temperature sensitivity in Escherichia coli. Microarray study revealed that the expression level of ybjN was inversely correlated with the expression of flagellar, fimbrial and acid resistance genes. Over-expression of ybjN significantly down-regulated genes involved in the citric acid cycle, glycolysis, the glyoxylate shunt, oxidative phosphorylation, and amino acid and nucleotide metabolism. On the other hand, over-expression of ybjN up-regulated toxin-antitoxin modules, the SOS responsive pathway, cold shock proteins and starvation-induced transporter genes. Our results collectively suggest that YbjN may play important roles in regulating bacterial multicellular behaviors, metabolism and survival under various stress conditions in Es. coli.