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 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 Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

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 Metabolic Engineering for l-Glutamine Overproduction by Using DNA Gyrase Mutations in Escherichia coli

2013 ◽  
Vol 79 (9) ◽  
pp. 3033-3039 ◽  
Author(s):  
Mikiro Hayashi ◽  
Kazuhiko Tabata
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Topoisomerase I ◽  
Dna Gyrase ◽  
Chromosomal Dna ◽  
Topoisomerase Iv ◽  
Α Subunit ◽  
Content Type ◽  
Genome Wide ◽  
K 12

ABSTRACTAnl-glutamine-overproducing mutant of anEscherichia coliK-12-derived strain was selected from randomly mutagenized cells in the course ofl-alanyl-l-glutamine strain development. Genome-wide mutation analysis unveiled a novel mechanism forl-glutamine overproduction in this mutant. Three mutations were identified that are related to thel-glutamine overproduction phenotype, namely, an intergenic mutation in the 5′-flanking region ofyeiGand two nonsynonymous mutations ingyrA(Gly821Ser and Asp830Asn). Expression ofyeiG, which encodes a putative esterase, was enhanced by the intergenic mutation. The nonsynonymous mutations ingyrA, a gene that encodes the DNA gyrase α subunit, affected the DNA topology of the cells. Gyrase is a type II topoisomerase that adds negative supercoils to double-stranded DNA. When the opposing DNA-relaxing activity was enhanced by overexpressing topoisomerase I (topA) and topoisomerase IV (parCandparE), an increase inl-glutamine production was observed. These results indicate that a reduction of chromosomal DNA supercoils in the mutant caused an increase inl-glutamine accumulation. The mechanism underlying this finding is discussed in this paper. We also constructed anl-glutamine-hyperproducing strain by attenuating cellularl-glutamine degradation activity. Although the reconstituted mutant (withyeiGtogether withgyrA) produced 200 mMl-glutamine, metabolic engineering finally enabled construction of a mutant that accumulated more than 500 mMl-glutamine.

scholarly journals Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Nicole Gruenheit ◽  
Amy Baldwin ◽  
Balint Stewart ◽  
Sarah Jaques ◽  
Thomas Keller ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
High Throughput ◽  
Gene Function ◽  
Evolutionary Biology ◽  
Large Scale ◽  
Function Analysis ◽  
Model Systems ◽  
Genome Wide ◽  
The Social ◽  
Drug Resistance Marker

Abstract Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. Results We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Conclusions We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

scholarly journals CS5 Pilus Biosynthesis Genes from Enterotoxigenic Escherichia coli O115:H40

1999 ◽  
Vol 181 (18) ◽  
pp. 5847-5851 ◽  
Author(s):  
Thomas G. Duthy ◽  
Lothar H. Staendner ◽  
Paul A. Manning ◽  
Michael W. Heuzenroeder
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Surface Expression ◽  
Open Reading Frames ◽  
Enterotoxigenic Escherichia Coli ◽  
Cell Surface Expression ◽  
E Coli ◽  
Pilus Biogenesis ◽  
K 12 ◽  
And Function

ABSTRACT We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designatedcsfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC,csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.

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