scholarly journals The essential genome ofEscherichia coliK-12

2017 ◽  
Author(s):  
Emily C. A. Goodall ◽  
Ashley Robinson ◽  
Iain G. Johnston ◽  
Sara Jabbari ◽  
Keith A. Turner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Statistical Analysis ◽  
Essential Gene ◽  
Automated Analysis ◽  
Transposon Mutagenesis ◽  
Essential Genes ◽  
High Density ◽  
Bacterial Physiology ◽  
E Coli ◽  
K 12

ABSTRACTTransposon-Directed Insertion-site Sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries and therefore it remains unclear whether the two methodologies are comparable. To address this, a high density transposon library was constructed inEscherichia coliK-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false positive identification of essential gene candidates, statistical data analysis included corrections for both gene length and genome length. Through this analysis new essential genes and genes previously incorrectly designated as essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects and fine resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis datasets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes inE. coli, we constructed a very high density transposon mutant library. Initial automated analysis of the resulting data revealed many discrepancies when compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high density TraDIS sequencing data for each putative essential gene for the model laboratory organism,Escherichia coli. This paper is important because it provides a better understanding of the essential genes ofE. coli, reveals the limitations of relying on automated analysis alone and a provides new standard for the analysis of TraDIS data.

scholarly journals The Essential Genome of Escherichia coli K-12

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Emily C. A. Goodall ◽  
Ashley Robinson ◽  
Iain G. Johnston ◽  
Sara Jabbari ◽  
Keith A. Turner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Statistical Analysis ◽  
Essential Gene ◽  
Automated Analysis ◽  
Transposon Mutagenesis ◽  
Essential Genes ◽  
High Density ◽  
Bacterial Physiology ◽  
E Coli ◽  
K 12

ABSTRACTTransposon-directed insertion site sequencing (TraDIS) is a high-throughput method coupling transposon mutagenesis with short-fragment DNA sequencing. It is commonly used to identify essential genes. Single gene deletion libraries are considered the gold standard for identifying essential genes. Currently, the TraDIS method has not been benchmarked against such libraries, and therefore, it remains unclear whether the two methodologies are comparable. To address this, a high-density transposon library was constructed inEscherichia coliK-12. Essential genes predicted from sequencing of this library were compared to existing essential gene databases. To decrease false-positive identification of essential genes, statistical data analysis included corrections for both gene length and genome length. Through this analysis, new essential genes and genes previously incorrectly designated essential were identified. We show that manual analysis of TraDIS data reveals novel features that would not have been detected by statistical analysis alone. Examples include short essential regions within genes, orientation-dependent effects, and fine-resolution identification of genome and protein features. Recognition of these insertion profiles in transposon mutagenesis data sets will assist genome annotation of less well characterized genomes and provides new insights into bacterial physiology and biochemistry.IMPORTANCEIncentives to define lists of genes that are essential for bacterial survival include the identification of potential targets for antibacterial drug development, genes required for rapid growth for exploitation in biotechnology, and discovery of new biochemical pathways. To identify essential genes inEscherichia coli, we constructed a transposon mutant library of unprecedented density. Initial automated analysis of the resulting data revealed many discrepancies compared to the literature. We now report more extensive statistical analysis supported by both literature searches and detailed inspection of high-density TraDIS sequencing data for each putative essential gene for theE. colimodel laboratory organism. This paper is important because it provides a better understanding of the essential genes ofE. coli, reveals the limitations of relying on automated analysis alone, and provides a new standard for the analysis of TraDIS data.

scholarly journals Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering

Microbiology ◽  
2014 ◽  
Vol 160 (11) ◽  
pp. 2341-2351 ◽  
Author(s):  
Mario Juhas ◽  
Daniel R. Reuß ◽  
Bingyao Zhu ◽  
Fabian M. Commichau
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Essential Gene ◽  
Building Blocks ◽  
Essential Genes ◽  
Minimal Cell ◽  
E Coli ◽  
Cell Factories ◽  
Gene Sets ◽  
K 12

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell ‘chassis’. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

scholarly journals Closed Genome Sequence of Escherichia coli K-12 Group Strain C600

2019 ◽  
Vol 8 (2) ◽  
Author(s):  
Anna Allué-Guardia ◽  
Emmanuel C. Nyong ◽  
Sara S. K. Koenig ◽  
Sean M. Vargas ◽  
James L. Bono ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Sequence ◽  
Laboratory Strain ◽  
Culture Collection ◽  
Content Type ◽  
Bacterial Physiology ◽  
E Coli ◽  
Molecular Microbiology ◽  
K 12 ◽  
Group Strain

Escherichia coli strain C600 is a prototypical K-12 derived laboratory strain which has been broadly used for molecular microbiology and bacterial physiology studies since its isolation in 1954. Here, we present the closed genome sequence of E. coli strain C600, retrieved from the American Type Culture Collection (ATCC 23724).

scholarly journals Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

2019 ◽  
Author(s):  
John S. Hawkins ◽  
Melanie R. Silvis ◽  
Byoung-Mo Koo ◽  
Jason M. Peters ◽  
Marco Jost ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cellular Networks ◽  
High Throughput ◽  
Essential Gene ◽  
Evolutionary Conservation ◽  
Essential Genes ◽  
E Coli ◽  
Gene Expression Levels

AbstractEssential genes are the central hubs of cellular networks. Despite their importance, the lack of high-throughput methods for titrating their expression has limited our understanding of the fitness landscapes against which essential gene expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of mismatched sgRNAs, we characterized the expression-fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. Remarkably, these relationships co-vary by pathway and are predominantly conserved between E. coli and B. subtilis despite ~ 2 billion years of evolutionary separation, suggesting that deeply conserved tradeoffs underlie bacterial homeostasis.One Sentence SummaryBacterial essential genes have varying responses to CRISPRi knockdown that are largely conserved across ~2 billion years of evolution.

scholarly journals Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability.

Genetics ◽  
1989 ◽  
Vol 122 (3) ◽  
pp. 491-501 ◽  
Author(s):  
B A Sampson ◽  
R Misra ◽  
S A Benson
Keyword(s):  
Escherichia Coli ◽  
Membrane Permeability ◽  
Outer Membrane ◽  
Essential Gene ◽  
Genetic Selection ◽  
E Coli ◽  
New Gene ◽  
Increased Sensitivity ◽  
K 12 ◽  
Outer Membrane Permeability

Abstract Using a genetic selection for mutations which allow large maltodextrins to cross the outer membrane of Escherichia coli in the absence of the LamB maltoporin, we have obtained and characterized two mutations that define a new locus of E. coli. We have designated this locus imp for increased membrane permeability. Mapping studies show that the imp gene resides at approximately 1.2 min on the E. coli chromosome. The mutations alter the permeability of the outer membrane resulting in increased sensitivity to detergents, antibiotics and dyes. The mutations are nonreverting and codominant. Genetic analysis of the mutations suggest that the imp gene is an essential gene. We describe a general cloning strategy that can be used to clone both dominant and recessive alleles. Using this technique, we have cloned the wild-type and mutant imp alleles onto a low copy number plasmid.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 81-88
Author(s):  
E H Berglin ◽  
M B Edlund ◽  
G K Nyberg ◽  
J Carlsson
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Metal Ion ◽  
Chelating Agent ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Anaerobic Conditions ◽  
Dna Breakage ◽  
E Coli ◽  
K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

scholarly journals The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6179-6185 ◽  
Author(s):  
Richard A. Lease ◽  
Dorie Smith ◽  
Kathleen McDonough ◽  
Marlene Belfort
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Resistance Genes ◽  
Acid Resistance ◽  
Dna Arrays ◽  
E Coli ◽  
Specific Mrnas ◽  
Steady State Levels ◽  
Control Translation ◽  
K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

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