Optimized protocols for ChIP-seq and deletion mutant construction in Pseudomonas syringae.
Ontology highlight
ABSTRACT:
SUBMITTER: Yao C
PROVIDER: S-EPMC8406033 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Pseudomonas syringae, a Gram-negative plant pathogen, infects more than 50 crops with its type III secretion system (T3SS) and causes severe economic losses around the world. Although the mechanisms of virulence-associated regulators of P. syringae T3SS have been studied for decades, the crosstalk and network underlying these regulators are still elusive. Previously, we have individually studied a group of T3SS regulators, including AefR, HrpS, and RhpRS. In the present study, we found 4 new T3SS regulator genes (envZ, ompR, tsiS and phoQ) via transposon-mediated mutagenesis. Two-component systems EnvZ and TsiS natively regulate T3SS. In order to uncover the crosstalk between 16 virulence-associated regulators, (including AefR, AlgU, CvsR, GacA, HrpL, HrpR, HrpS, MgrA, OmpR, PhoP, PilR, PsrA, RhpR, RpoN, TsiR and Vfr) in P. syringae, we mapped an intricate network named PSVnet (Pseudomonas syringae Virulence Regulatory Network) by combining differentially expression genes in RNA-seq and binding loci in ChIP-seq of all regulators.
2021-03-29 | GSE138204 | GEO
Project description:Chromatin immunoprecipitation (ChIP)-seq analyses of transcription factors in clinical specimens are challenging due to the technical limitations and low quantities of starting material, often resulting in low enrichments and poor signal-to-noise ratio. Here, we present an optimized protocol for transcription factor ChIP-seq analyses in human tissue, yielding an ?100% success rate for all transcription factors analyzed. As proof of concept and to illustrate general applicability of the approach, human tissue from the breast, prostate, and endometrial cancers were analyzed. In addition to standard formaldehyde fixation, disuccinimidyl glutarate was included in the procedure, greatly increasing data quality. To illustrate the sensitivity of the optimized protocol, we provide high-quality ChIP-seq data for three independent factors (AR, FOXA1, and H3K27ac) from a single core needle prostate cancer biopsy specimen. In summary, double-cross-linking strongly improved transcription factor ChIP-seq quality on human tumor samples, further facilitating and enhancing translational research on limited amounts of tissue.
| S-EPMC6311467 | biostudies-literature
Project description:We developed a computational procedure for optimizing the binding site detections in a given ChIP-seq experiment by maximizing their reproducibility under bootstrap sampling. We demonstrate how the procedure can improve the detection accuracies beyond those obtained with the default settings of popular peak calling software, or inform the user whether the peak detection results are compromised, circumventing the need for arbitrary re-iterative peak calling under varying parameter settings. The generic, open-source implementation is easily extendable to accommodate additional features and to promote its widespread application in future ChIP-seq studies. The peakROTS R-package and user guide are freely available at http://www.nic.funet.fi/pub/sci/molbio/peakROTS.
| S-EPMC3245948 | biostudies-literature
Project description:The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.
| S-EPMC4886819 | biostudies-literature
Project description:Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a.
| S-EPMC3329529 | biostudies-literature
Project description:Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing is a powerful technique that characterizes the genome-wide DNA-binding profile of a protein of interest. The general ChIP-seq workflow has been applied widely to many sample types and target proteins, but sample-specific optimization of various steps is necessary to achieve high-quality data. This protocol is specifically optimized for cultured human embryonic stem cells (hESCs), including steps to check sample quality and non-specific enrichment of "hyper-ChIPable" regions prior to sequencing. For complete details on the use and execution of this protocol, please refer to Gunne-Braden et al. (2020).
| S-EPMC7501726 | biostudies-literature
Project description:OBJECTIVE:To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination. METHODS:First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create up?-down amplicon. Then, the up?-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe. RESULTS:The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly. CONCLUSIONS:The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.
| S-EPMC7041158 | biostudies-literature
Project description:The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high-quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein cross-linking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.
| S-EPMC8961045 | biostudies-literature
Project description:Bacteria relies on two-component systems (TCSs) to respond to a wide range of stimuli or environmental cues for their survival and virulence. However, the functions and synergistic actions of TCSs in genomic level remains unclear. Here, in model phytopathogen Pseudomonas syringae, by integrating multiomics data, we developed a network-based PSTCSome (Pseudomonas syringae two-component systems regulome) to identify functions and crosstalk among global TCSs under either virulence suppressing (King’s B medium, KB) or activating conditions (minimal medium, MM). Transcriptome profiling identifies 2,099 differentially expressed genes (DEGs) in KB and 1,250 DEGs in MM, while ChIP-seq identifies 1,628 target genes across the whole genome. The multiomics results are applied to perform not only gene ontology (GO) analysis to detect the biological processes that TCSs involved in, but also subnetworks and co-expression analysis of 8 virulence-related pathways to decipher the TCSs regulated pathogenic behaviors. The following phenotypic experiments newly confirmed 8 TCSs that regulates type III secretion system (T3SS) (PSPPH_0253, PSPPH_2606, PSPPH_4416, and PSPPH_4451) and surface attachment (PSPPH_0253, PSPPH_3041, PSPPH_3473, PSPPH_3736, PSPPH_4001). We then compute 259 functional genes in KB and 161 in MM for those cluster TCSs. Analysis of cluster TCSs regulons led to the identification of either novel functions or 2 master regulatory TCSs (RhpS/RhpR and PSPPH_4827/4828) toward virulence. Our results show that TCSs in P. syringae dynamically adjust their regulatory networks by sensing the external environment, and then switch bacteria between pathogenic and non-pathogenic states in a sophisticated way. Furthermore, we present an online platform of PSTCSome to facilitate updating, network visualization and user-customized analyses.
2022-10-04 | GSE148104 | GEO
Project description:In order to better understand the transcriptional networks triggered by pathogen inoculation, we monitored gene expression in leaves of mutant Arabidopsis plants, inoculated with Pseudomonas syringae ES4326 and wild type Col-0 plants grown in parallel. Individual leaves were injected in the morning using a needle-less syringe with 10E5 cfu cm-2 PsmES4326 (suspended in 5 mM MgSO4). For the wild type, leaves were also mock treated with 5 mM MgSO4. Leaves were harvested 24 hours later. Plants were grown in pots with BM-2 soil (Berger Peat Moss Ltd, Quebec, Canada) at a density of 9 plants per pot and kept at 22 degrees Celsius with 75% humidity and a 12 hour day length. Keywords: Expression profilling by array
2009-11-18 | GSE18978 | GEO