scholarly journals Self-immunity guided identification of threonyl-tRNA synthetase as the molecular target of obafluorin, a β-lactone antibiotic

2019 ◽  
Author(s):  
Thomas A. Scott ◽  
Sibyl F. Batey ◽  
Patrick Wiencek ◽  
Govind Chandra ◽  
Silke Alt ◽  
...  
Keyword(s):  
Drug Targets ◽  
De Novo ◽  
Molecular Target ◽  
Trna Synthetase ◽  
Inhibition Mechanism ◽  
Enzyme Assays ◽  
E Coli ◽  
Biological Target ◽  
Antibiotic Discovery ◽  
Chemical Matter

AbstractTo meet the ever-growing demand of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique β-lactone obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Grampositive and -negative pathogens, however the biological target was unknown. We now report that obafluorin targets threonyl-tRNA-synthetase and we identify a homologue, ObaO, which confers self-immunity to the obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of a self-immunity guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved obafluorin variants.

Substituted 6,7-dimethoxy-5-oxo-2,3,5,9b-tetrahydrothiazolo[2,3-a]isoindole- 3-1,1-dioxide Derivatives with Antimicrobial Activity and Docking Assisted Prediction of the Mechanism of their Antibacterial and Antifungal Properties

2020 ◽  
Vol 20 (29) ◽  
pp. 2681-2691
Author(s):  
Athina Geronikaki ◽  
Victor Kartsev ◽  
Phaedra Eleftheriou ◽  
Anthi Petrou ◽  
Jasmina Glamočlija ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antifungal Activity ◽  
Drug Targets ◽  
Bacterial Species ◽  
Pharmaceutical Research ◽  
Docking Studies ◽  
Fungal Species ◽  
Docking Analysis ◽  
E Coli ◽  
Antibacterial And Antifungal

Background: Although a great number of the targets of antimicrobial therapy have been achieved, it remains among the first fields of pharmaceutical research, mainly because of the development of resistant strains. Docking analysis may be an important tool in the research for the development of more effective agents against specific drug targets or multi-target agents 1-3. Methods: In the present study, based on docking analysis, ten tetrahydrothiazolo[2,3-a]isoindole derivatives were chosen for the evaluation of the antimicrobial activity. Results: All compounds showed antibacterial activity against eight Gram-positive and Gram-negative bacterial species being, in some cases, more potent than ampicillin and streptomycin against all species. The most sensitive bacteria appeared to be S. aureus and En. Cloacae, while M. flavus, E. coli and P. aeruginosa were the most resistant ones. The compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited good antifungal activity better than reference drugs bifonazole (1.4 – 41 folds) and ketoconazole (1.1 – 406 folds) against all fungal species. In order to elucidate the mechanism of action, docking studies on different antimicrobial targets were performed. Conclusion: According to docking analysis, the antifungal activity can be explained by the inhibition of the CYP51 enzyme for most compounds with a better correlation of the results obtained for the P.v.c. strain (linear regression between estimated binding Energy and log(1/MIC) with R 2 =0.867 and p=0.000091 or R 2 = 0.924, p= 0.000036, when compound 3 is excluded.

scholarly journals 4E Interacting Protein as a Potential Novel Drug Target for Nucleoside Analogues in Trypanosoma Brucei

2021 ◽  
Vol 9 (4) ◽  
pp. 826
Author(s):  
Dorien Mabille ◽  
Camila Cardoso Santos ◽  
Rik Hendrickx ◽  
Mathieu Claes ◽  
Peter Takac ◽  
...  
Keyword(s):  
Mode Of Action ◽  
Drug Target ◽  
Drug Targets ◽  
De Novo ◽  
Treatment Options ◽  
Adenosine Kinase ◽  
Nucleoside Analogues ◽  
Purine Salvage ◽  
Interacting Protein ◽  
Novel Drug

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets. The activity of known nucleoside analogues such as tubercidin and cordycepin led to the development of a series of C7-substituted nucleoside analogues. Here, we use RNA interference (RNAi) libraries to gain insight into the mode-of-action of these novel nucleoside analogues. Whole-genome RNAi screening revealed the involvement of adenosine kinase and 4E interacting protein into the mode-of-action of certain antitrypanosomal nucleoside analogues. Using RNAi lines and gene-deficient parasites, 4E interacting protein was found to be essential for parasite growth and infectivity in the vertebrate host. The essential nature of this gene product and involvement in the activity of certain nucleoside analogues indicates that it represents a potential novel drug target.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

2012 ◽  
Vol 78 (20) ◽  
pp. 7407-7413 ◽  
Author(s):  
Qian Zhang ◽  
Tao Yan
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Potassium Acetate ◽  
Desiccation Stress ◽  
Desiccation Resistance ◽  
Content Type ◽  
Soil Desiccation ◽  
E Coli ◽  
Organic Solute ◽  
Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution

Science ◽  
1989 ◽  
Vol 246 (4934) ◽  
pp. 1135-1142 ◽  
Author(s):  
M. Rould ◽  
J. Perona ◽  
D Soll ◽  
T. Steitz
Keyword(s):  
Trna Synthetase ◽  
E Coli

scholarly journals Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Zhenning Liu ◽  
Xue Zhang ◽  
Dengwei Lei ◽  
Bin Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
De Novo ◽  
Microbial Cell ◽  
Cell Factory ◽  
Phosphopantetheinyl Transferase ◽  
Chromosome Engineering ◽  
Microbial Cell Factory ◽  
E Coli ◽  
Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Proteomic analysis of the colistin-resistant E. coli clinical isolate: Explorations of the resistome

2021 ◽  
Vol 28 ◽  
Author(s):  
Divakar Sharma ◽  
Manisha Aswal ◽  
Nayeem Ahmad ◽  
Manish Kumar ◽  
Asad U Khan
Keyword(s):  
Clinical Isolate ◽  
Protein Interaction ◽  
Functional Annotation ◽  
Drug Targets ◽  
Bacterial Infections ◽  
Sugar Metabolism ◽  
Colistin Resistance ◽  
Phosphotransferase System ◽  
E Coli ◽  
Protein Protein Interaction

Background: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. Objective: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. Methods: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. Results: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. Conclusion: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

Sign in / Sign up

Export Citation Format

Share Document