scholarly journals Genome-wide high-throughput screening of interactive bacterial metabolite in the algal population using Escherichia coli K-12 Keio collection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jina Heo ◽  
Kichul Cho ◽  
Urim Kim ◽  
Dae-Hyun Cho ◽  
Sora Ko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Algal Population ◽  
Genome Wide ◽  
Keio Collection ◽  
K 12

scholarly journals An Escherichia coli Biosensor Strain for Amplified and High Throughput Detection of Antimicrobial Agents

2002 ◽  
Vol 7 (2) ◽  
pp. 119-125 ◽  
Author(s):  
Minna-Liisa Anko ◽  
Jussi Kurittu ◽  
Matti Karp
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Agents ◽  
Test Organism ◽  
Plasmid Copy Number ◽  
Host Cells ◽  
Reporter System ◽  
Plasmid Copy ◽  
K 12

We report here the construction of a bacterial reporter system for high-throughput screening of antimicrobial agents. The test organism is the Escherichia coli K-12 strain carrying luciferase genes luxC, luxD, luxA, luxB, and luxE from the bioluminescent bacterium Photorhabdus luminescens in a runaway replication plasmid. The replication of the plasmid can be induced, resulting in a change of the plasmid copy number from 1-2/cell to several hundreds per cell within tens of minutes. This increase in plasmid copies is independent of the replication of the host cells. The system will therefore amplify the effects of antibiotics inhibiting bacterial replication machinery, such as fluoroquinolones, and the inhibitory effects can be measured in real time by luminometry. The biosensor was compared with a strain engineered to emit light constitutively, and it was shown to be much more sensitive to various antibiotics than conventional overnight cultivation methods. The approach shows great potential for high-throughput screening of new compounds.

scholarly journals Insights from the reanalysis of high-throughput chemical genomics data for Escherichia coli K-12

2020 ◽  
Author(s):  
Peter I-Fan Wu ◽  
Curtis Ross ◽  
Deborah A. Siegele ◽  
James C. Hu
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Gene Function ◽  
Chemical Genomics ◽  
Phenotypic Data ◽  
Phenotypic Profile ◽  
Functional Connections ◽  
Genome Wide ◽  
K 12 ◽  
And Function

ABSTRACTDespite the demonstrated success of genome-wide genetic screens and chemical genomics studies at predicting functions for genes of unknown function or predicting new functions for well-characterized genes, their potential to provide insights into gene function hasn’t been fully explored. We systematically reanalyzed a published high-throughput phenotypic dataset for the model Gram-negative bacterium Escherichia coli K-12. The availability of high-quality annotation sets allowed us to compare the power of different metrics for measuring phenotypic profile similarity to correctly infer gene function. We conclude that there is no single best method; the three metrics tested gave comparable results for most gene pairs. We also assessed how converting qualitative phenotypes to discrete, qualitative phenotypes affected the association between phenotype and function. Our results indicate that this approach may allow phenotypic data from different studies to be combined to produce a larger dataset that may reveal functional connections between genes not detected in individual studies.

scholarly journals Insights from the reanalysis of high-throughput chemical genomics data for Escherichia coli K-12

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter I-Fan Wu ◽  
Curtis Ross ◽  
Deborah A Siegele ◽  
James C Hu
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Gene Function ◽  
Chemical Genomics ◽  
Phenotypic Data ◽  
Phenotypic Profile ◽  
Functional Connections ◽  
Genome Wide ◽  
K 12 ◽  
And Function

Abstract Despite the demonstrated success of genome-wide genetic screens and chemical genomics studies at predicting functions for genes of unknown function or predicting new functions for well-characterized genes, their potential to provide insights into gene function has not been fully explored. We systematically reanalyzed a published high-throughput phenotypic dataset for the model Gram-negative bacterium Escherichia coli K-12. The availability of high-quality annotation sets allowed us to compare the power of different metrics for measuring phenotypic profile similarity to correctly infer gene function. We conclude that there is no single best method; the three metrics tested gave comparable results for most gene pairs. We also assessed how converting quantitative phenotypes to discrete, qualitative phenotypes affected the association between phenotype and function. Our results indicate that this approach may allow phenotypic data from different studies to be combined to produce a larger dataset that may reveal functional connections between genes not detected in individual studies.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

scholarly journals Fluorescence Detection-Based Functional Assay for High-Throughput Screening for MraY

2004 ◽  
Vol 48 (3) ◽  
pp. 897-902 ◽  
Author(s):  
Thérèse Stachyra ◽  
Christophe Dini ◽  
Paul Ferrari ◽  
Ahmed Bouhss ◽  
Jean van Heijenoort ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Pressure ◽  
Cell Wall ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reaction Medium ◽  
The Other ◽  
Functional Assay ◽  
Liquid Chromatography Separation ◽  
Fluorescent Derivative

ABSTRACT We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-Nε -dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

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