Journal: The EMBO Journal

Article Title: BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses

doi: 10.15252/embj.2018100293

Figure Lengend Snippet:

Article Snippet: Second‐strand cDNAs were synthesized using E. coli DNA polymerase I and RNaseH (Invitrogen).

Techniques: Recombinant, Positive Control, Microarray, RNA Sequencing, ChIP-sequencing

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: ( a ) Schematic of the ‘clip-chip’ strategy. ( b,c ) Identification of YcgC as a potential protein deacetylase. E. coli proteome chips were clipped onto three substrate slides separately coated with acetylated RutR, NhoA, and YceC. After incubation in a protein deacetylase buffer, the reactions were terminated by adding wash buffers, followed by a signal detection step with a pan α-AcK antibody coupled with a Cy3-labeled secondary antibody as detection reagent to visualize the loss of signals (e.g., black holes in ( b,c ). To determine the identity of proteins that generated the holes, the substrate slide was subsequently probed with an α-6xHis antibody followed by a Cy5-conjugated secondary antibody. ( d ) Using acetylated RutR proteins purified from E. coli , of the four candidates tested, YcgC showed robust deacetylation activity in vitro. Equal amounts of RutR proteins were used in each reaction and loss of acetylation was detected with the pan α-AcK antibody. DOI: http://dx.doi.org/10.7554/eLife.05322.003

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: Histone Deacetylase Assay, Incubation, Labeling, Generated, Purification, Activity Assay, In Vitro

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: ( a ) In vitro assays of the KDAC activity of YcgC on RutR demonstrated that its KDAC activity does not require either NAD + or Zn 2+ as cofactors. Incubation with YcgC almost completely abolished the slower migrating acetylated RutR bands (upper panel) as evidenced by immonublotting (lower panel). ( b,c ) LC-MS/MS analysis to determine the residues of RutR deacetylated by YcgC. RutR was treated with YcgC first and the untreated RutR used as the control. Both these two samples were resolved on a SDS-PAGE gel side by side. The upper band represents the Kac-containing starting materials and the lower band represents the K-containing product, which were then recovered from the gel and subjected for MS/MS analysis (inserts). Lys52 was identified as an acetylated site in RutR protein ( b ). After incubating with YcgC, acetylation on K52 was no longer detectable ( c ). ( d ) RutR is deacetylated by YcgC in Escherichia coli . A 3xFLAG tag was chromosomally inserted at the 3′-end of rutR coding sequence. Acetylation of 3xFLAG-tagged RutR was monitored upon induction of YcgC. While the protein level of RutR was unchanged (middle panel), its acetylation level was dramatically reduced as a function of YcgG induction (upper panel). YcgC’s expression was monitored using a custom-made antibody (lower panel). ( e ) Mutagenesis of RutR confirmed that K52 and K62 are acetylated in vivo. Two single mutants K52Q and K62Q and one double mutant K52/62Q were constructed. These mutants along with WT RutR were produced and purified in E. coli . Equal amounts of purified proteins were Western blotted with the α-AcK antibody, quantitation of acetylation level of these samples were performed. KDAC: Lysine deacetylase; LC-MS/MS: Liquid chromatography–mass spectrometry; IP: Immunoprecipitation. DOI: http://dx.doi.org/10.7554/eLife.05322.005

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: In Vitro, Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy, Control, SDS Page, Tandem Mass Spectroscopy, Sequencing, Expressing, Mutagenesis, In Vivo, Construct, Produced, Purification, Western Blot, Quantitation Assay, Histone Deacetylase Assay, Liquid Chromatography, Mass Spectrometry, Immunoprecipitation

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: Specificity was measured by spiking an E. coli total lysate with affinity purified YcgC, and sensitivity by testing serially diluted YcgC. DOI: http://dx.doi.org/10.7554/eLife.05322.008

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: Affinity Purification

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: Four single mutants K52Q, K62Q, K52R, and K62R and two double mutants K52/62Q and K52/62R were constructed. These mutants along with WT RutR were produced and purified in E. coli . Equal amounts of purified proteins were Western blotted with the α-AcK antibody. DOI: http://dx.doi.org/10.7554/eLife.05322.009

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: Construct, Produced, Purification, Western Blot

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: ( a ) YcgC regulates gene expression via deacetylating RutR. Expression of gcd and pmrD is significantly reduced upon RutR induction over a period of 2 hr as measured by quantitative real-time PCR. Double asterisks indicate that the observed fold changes are statistically significant, p<0.01. ( b ) Global gene expression analysis of ycgC - and cobB -induced cells. Clustering analysis shows clearly that impact on global transcription of induction of ycgC is distinct from that of cobB . Venn diagram showing that there is no significant overlap between genes down- and up-regulated due to CobB and YcgC induction. ( c ) Overexpression of YcgC affects global protein acetylation levels in E. coli . After ycgC and cobB were separately induced for 1 hr, global acetylation was detected in whole lysates of Escherichia coli using two pan α-AcK antibodies. The WT E. coli strain was also included for comparison. Boxed areas indicate regions that show obviously different staining patterns in ycgC - and cobB -induced cells. PCR: Polymerase chain reaction; WT: Wild type. DOI: http://dx.doi.org/10.7554/eLife.05322.014

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Over Expression, Comparison, Staining, Polymerase Chain Reaction

Journal: eLife

Article Title: YcgC represents a new protein deacetylase family in prokaryotes

doi: 10.7554/eLife.05322

Figure Lengend Snippet: ( a ) Five representative YcgC homologs with protein sequence homology ranging from low to high. ( b ) Amino acid sequence homology analysis between YcgC and five selected YcgC homologs from other bacteria. The consensus strength among the six homologous proteins at each amino acid position of YcgC is indicated with colored bars. Red, orange, green, light blue, dark blue, and blank bars represent 100, 80, 60, 40, 20, and 0% consensus strength, respectively. ( c,d ) Changes in global E. coli acetylation profiles upon induction of the five YcgC homologs. The five selected YcgC homologs were cloned, transformed into E. coli , and induced to overexpress. Global acetylation profiles of each induced strain were detected with a pan monoclonal antibody (Cell Signaling, #9441) and a pan polyclonal antibody (PTM-Biolabs, PTM-105), as shown in c and d , respectively. WT E. coli cells were also processed in parallel as a comparison. An antibody against myelin basic protein was used as a loading control. WT: Wild type. DOI: http://dx.doi.org/10.7554/eLife.05322.015

Article Snippet: E. coli DNA chips were purchased from CapitalBio Corp. (Beijing, China). cDNA labeled with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine’s linear RNA amplification method and subsequent enzymatic reaction ( ; ).

Techniques: Sequencing, Bacteria, Clone Assay, Transformation Assay, Comparison, Control

Journal: PLoS Genetics

Article Title: A Comprehensive Genetic Characterization of Bacterial Motility

doi: 10.1371/journal.pgen.0030154

Figure Lengend Snippet: (A) Swarming behavior of the Δ yhjH mutant. (B) Software-assisted tracking of tethered cells. The rotational direction of single motors were followed in time. CCW direction (swimming) is designated by 1 and CW direction (tumbling) is indicated by a −1. Wild-type cells switched frequently between the swimming and tumbling motor states while Δ cheR and Δ yhjH remained mostly in the swimming state. Averages and standard error are as follows: wild type, 0.32 ± 0.18, n = 28; Δ yhjH, 0.88 ± 0.10, n = 22; Δ cheR, 0.99 ± 0.03, n = 23; Δ cheZ, −0.99 ± 0.01, n = 23. (C) Soft agar was inoculated with Δ yhjH mutants (left) and with a library of transposon insertional double mutants (right). After a 20-h incubation at 30 °C, mutants that showed a suppression of the motility defect were extracted from the agar (red dashed line) and used for further analyses. (D) The ycgR transcriptional unit is shown. Ten insertional mutations were mapped to nine different locations in the ycgR gene. Triangles denote the sites of the insertions; strain names appear next to each insertion site. Triangles above the gene indicate transposon insertions with the T7 promoter oriented to the right, and triangles below represent the promoter facing the left. Transcription of ycgR is positively regulated by FlhDC . (E) Expression of yfgF partly suppresses the motility defect of the Δ yhjH mutant. Wild-type and mutant strains were transformed with empty pBAD18 or with pBAD18 carrying yfgF cloned in front of the arabinose-inducible promoter. Overnight cultures were used to inoculate soft agar plates containing 100 μg/ml ampicillin and 0.2% arabinose. Images were taken after 8 h at 30 °C. (F) Double mutants were generated (as described in ) based on microarray results. Displayed are mutants that show partial suppression of the Δ yhjH motility defect. (G) Domain organization of YhjH, YfgF, and YegE with predicted biochemical activities in E. coli . Domain structure is diagrammed according to Pfam ( http://www.sanger.ac.uk/Software/Pfam ). PDE, phosphodiesterase; DGC, diguanylate cyclase; PAS, ubiquitous signal sensor domain ; MASE1, membrane-associated sensor domain ; GMP, guanosine monophosphate; and GTP, guanosine triphosphate.

Article Snippet: Ligation products were purified using QIAquick gel extraction columns (Qiagen, http://www1.qiagen.com/ ), treated with E. coli DNA polymerase I (New England Biolabs, http://www.neb.com/ ), and used as template in a PCR containing a primer specific to the transposon and a primer specific to the Y-shaped linker, 0.2 mM dNTP mix, PCR buffer, and ExTaq DNA polymerase (TaKaRa, http://www.takara-bio.com/ ).

Techniques: Mutagenesis, Software, Incubation, Expressing, Transformation Assay, Clone Assay, Generated, Microarray

Journal:

Article Title: DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate

doi: 10.1128/AEM.69.3.1759-1774.2003

Figure Lengend Snippet: Hierarchical cluster analysis of gene expression changes in response to sodium acetate, sodium propionate, sodium chloride, and CCCP. Gene expression data from 27 microarray experiments (columns) and 179 genes (lines) are represented. Microarray experiments involved comparing gene expression by E. coli MG1655 on LB medium (reference) to gene expression 5 min (Ac_pulse_#I and II) and 50 min (Ac_50 min_#I to VI) after the addition of acetate, after adaptation to acetate (Ac_adapt_#I to IV), 5 min after the addition of propionate (Prop_pulse), after adaptation to propionate (Prop_adapt_#I to III), after adaptation to increased sodium chloride concentrations (NaCl_#I to III), and 5 min (CCCP_pulse_#I and II) and 3 h (CCCP_adapt_#I and II) after the addition of CCCP. Gene expression after the adaptation of MG1655 to acetate was compared directly to that after adaptation to propionate (Ac_vs_Prop_adapt_#I to III). Gene expression by the lrhA disruption strain IMW331 on LB medium (reference) was compared to gene expression after adaptation to acetate (Ac_adapt_delta_LrhA). Green indicates decreased RNA levels and red indicates increased RNA levels in the test culture compared to the reference culture. (A) Graph depicting the whole cluster (see the text for details). Subclusters 1 and 2 are highlighted in red, and subcluster 3 is highlighted in green. The scale bar indicates color coding of the RNA levels. (B to D) Subclusters 1 to 3, respectively, from panel A with corresponding gene names.

Article Snippet: Fluorescence-labeled DNA derived from random priming of 2 μg of E. coli genomic DNA with the large fragment of DNA polymerase I (Gibco BRL, Life Technologies) was hybridized.

Techniques: Expressing, Microarray

Journal:

Article Title: DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate

doi: 10.1128/AEM.69.3.1759-1774.2003

Figure Lengend Snippet: Relative RNA levels for genes in DNA microarray comparisons of global gene expression in E. coli MG1655 grown on various media a

Article Snippet: Fluorescence-labeled DNA derived from random priming of 2 μg of E. coli genomic DNA with the large fragment of DNA polymerase I (Gibco BRL, Life Technologies) was hybridized.

Techniques: Microarray, Expressing, Binding Assay, Blocking Assay, Chemotaxis Assay, Diffusion-based Assay

Journal:

Article Title: DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate

doi: 10.1128/AEM.69.3.1759-1774.2003

Figure Lengend Snippet: Expression of chromosomal fliC ′-′ lacZ and flhDC ′-′ lacZ translational fusions in E. coli IMW356 and CP992, respectively, and mRNA levels in E. coli MG1655 grown for ∼20 generations on various media a

Article Snippet: Fluorescence-labeled DNA derived from random priming of 2 μg of E. coli genomic DNA with the large fragment of DNA polymerase I (Gibco BRL, Life Technologies) was hybridized.

Techniques: Expressing, Activity Assay

Journal:

Article Title: DNA Microarray Analyses of the Long-Term Adaptive Response of Escherichia coli to Acetate and Propionate

doi: 10.1128/AEM.69.3.1759-1774.2003

Figure Lengend Snippet: Relative RNA levels of genes in DNA microarray comparisons of global gene expression in E. coli MG1655 cells grown for ∼20 generations on various media a

Article Snippet: Fluorescence-labeled DNA derived from random priming of 2 μg of E. coli genomic DNA with the large fragment of DNA polymerase I (Gibco BRL, Life Technologies) was hybridized.

Techniques: Microarray, Expressing

Journal: Cell reports

Article Title: Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function

doi: 10.1016/j.celrep.2018.05.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cricket paralysis virus stocks (CrPV) were kindly provided by Eric Jan. Escherichia coli strain K-12 (ATCC) was grown in Luria-Bertani, Miller broth (ThermoFisher).

Techniques: Virus, Recombinant, Luciferase, Microarray, Gene Expression, Retroviral, Control, Mutagenesis, Radial Immuno Diffusion, CRISPR, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Software

Journal: Infection and Immunity

Article Title: Transcriptome Analysis of Avian Pathogenic Escherichia coli O1 in Chicken Serum Reveals Adaptive Responses to Systemic Infection ^▿

doi: 10.1128/IAI.01230-10

Figure Lengend Snippet: Comparison of gene regulation analyzed by microarray (filled) or real-time quantitative RT-PCR (open). Real-time quantitative RT-PCR was used to validate the expression level for 11 selected genes, including 6 upregulated genes and 4 downregulated genes revealed by microarray analysis.

Article Snippet: The 8 × 15,000 (15K) DNA high-density microarray of APEC O1 was designed by Oxford Gene Technology (Oxford OX5 1PF, United Kingdom) and validated by the University of Birmingham E. coli Centre (UBEC) (United Kingdom).

Techniques: Comparison, Microarray, Quantitative RT-PCR, Expressing