scholarly journals Escherichia colimazEF Toxin-Antitoxin System as a Tool to Target Cell Ablation in Plants

2016 ◽  
Vol 26 (4) ◽  
pp. 277-283 ◽  
Author(s):  
Fabien Baldacci-Cresp ◽  
Anaxi Houbaert ◽  
Amana Metuor Dabire ◽  
Adeline Mol ◽  
Daniel Monteyne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Plant Cell ◽  
Target Cell ◽  
Cell Layer ◽  
Tapetal Cell ◽  
Plant Cells ◽  
Cell Types ◽  
E Coli ◽  
Cell Ablation

<b><i>Background/Aims:</i></b> The <i>Escherichia coli</i> MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the <i>mazEF</i> toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation. <b><i>Methods:</i></b> A double transformation strategy, combining expression of the <i>mazE</i> antitoxin gene under the control of the <i>CaMV 35S</i> promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the <i>mazF </i>gene under the control of the <i>TA29</i> tapetum-specific promoter in transgenic tobacco, was applied. <b><i>Results:</i></b> No transgenic <i>TA29-mazF</i> line could be regenerated, suggesting that the <i>TA29</i> promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated<i> 35S-mazE</i>/<i>TA29-mazF</i> double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen. <b><i>Conclusion:</i></b> These results show that the <i>E. coli</i><i>mazEF</i> system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.

ESCHERICHIA COLI DIE-OUT FROM NORMAL SOIL AS RELATED TO NUTRIENT AVAILABILITY AND THE INDIGENOUS MICROFLORA

1967 ◽  
Vol 13 (11) ◽  
pp. 1461-1470 ◽  
Author(s):  
D. A. Klein ◽  
L. E. Casida Jr.
Keyword(s):  
Escherichia Coli ◽  
Organic Carbon ◽  
Cell Types ◽  
Natural Soil ◽  
E Coli ◽  
Degree Of Protection ◽  
Normal Soil ◽  
Micro Organisms ◽  
Effect Of Glucose ◽  
Amended Soil

Addition of a small portion of autoclaved soil to normal soil provided some degree of protection against die-out for Escherichia coli cells also introduced into this soil. This protection was associated with organic carbon (glucose) availability, but not with nitrogen (ammonium nitrate) availability. An E. coli population was stabilized, at least temporarily, in soil by glucose feeding, and the protective effect of glucose was temperature dependent. Indigenous micro-organisms also multiplied in the glucose-amended soil, and, therefore, colonizing space and microbial production of compounds toxic to E. coli were not considered to be major controlling factors in E. coli die-out in soil. E. coli cells recovered after residence in this soil demonstrated an initial decreased growth rate which was less pronounced in cells recovered from glucose-amended soil and which disappeared for both cell types on subculture. It was concluded that a major factor in E. coli die-out from natural soil is its inability to step down its metabolic rate to meet the low availability of usable organic carbon in soil.

scholarly journals The Action of Bacteriophage Ω8 on Two Strains of Escherichia coli 08

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Barbara Wallenfels ◽  
K. Jann
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Phage Particle ◽  
Cytoplasmic Membrane ◽  
Cellular Respiration ◽  
Bacterial Strains ◽  
Viral Dna ◽  
Killing Effect ◽  
E Coli ◽  
Phage Propagation

Bacteriophage Ω8 is propagated in Escherichia coli E56b (08: K27-:H-), a non-capsulated strain. Another non-capsulated strain, E. coli 2398 (08:K?-:H-), is killed by bacteriophage Ω8 without phage propagation. This strain was formerly believed to be E. coli 093:K?-:H-, cross-reacting with strain E56b. We have established chemical and serological identity of the 08-specific lipopolysaccharides of the two strains. The 08-specific lipopolysaccharides of both strains inhibited the infection of Escherichia coli E56b with bacteriophage Ω8 equally well. The adsorption rate constants of Ω8 were identical for the two strains of E. coli 08. Evidence was obtained with 32P-labelled bacteriophage Ω8 for penetration of viral DNA into both bacterial strains. In host strain E56b, phage particle synthesis occurred normally. In strain 2398 the viral DNA was not degraded but its expression was blocked. The killing effect of Ω8 on E. coli strain 2398 is supposed to be due to damage of the cytoplasmic membrane, which could not be reversed under the influence of viral information. This was indicated by a blockage of cellular respiration, β-galactoside transport and RNA as well as protein synthesis.

scholarly journals From reporters to endogenous genes: the impact of the first five codons on translation efficiency in Escherichia coli

2019 ◽  
Author(s):  
Mariana H. Moreira ◽  
Géssica C. Barros ◽  
Rodrigo D. Requião ◽  
Silvana Rossetto ◽  
Tatiana Domitrovic ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Protein Expression ◽  
Nucleotide Composition ◽  
Control Mechanisms ◽  
Translation Efficiency ◽  
Coding Region ◽  
E Coli ◽  
Endogenous Proteins ◽  
The Impact

ABSTRACTTranslation initiation is a critical step in the regulation of protein synthesis, and it is subjected to different control mechanisms, such as 5’ UTR secondary structure and initiation codon context, that can influence the rates at which initiation and consequentially translation occur. For some genes, translation elongation also affects the rate of protein synthesis. With a GFP library containing nearly all possible combinations of nucleotides from the 3rd to the 5th codon positions in the protein coding region of the mRNA, it was previously demonstrated that some nucleotide combinations increased GFP expression up to four orders of magnitude. While it is clear that the codon region from positions 3 to 5 can influence protein expression levels of artificial constructs, its impact on endogenous proteins is still unknown. Through bioinformatics analysis, we identified the nucleotide combinations of the GFP library in Escherichia coli genes and examined the correlation between the expected levels of translation according to the GFP data with the experimental measures of protein expression. We observed that E. coli genes were enriched with the nucleotide compositions that enhanced protein expression in the GFP library, but surprisingly, it seemed to affect the translation efficiency only marginally. Nevertheless, our data indicate that different enterobacteria present similar nucleotide composition enrichment as E. coli, suggesting an evolutionary pressure towards the conservation of short translational enhancer sequences.

scholarly journals Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

Microtubules in plant cell division

1989 ◽  
Vol 47 ◽  
pp. 758-759
Author(s):  
S. M. Wick
Keyword(s):  
Electron Microscopy ◽  
Plant Cell ◽  
Mother Cell ◽  
Immunofluorescence Microscopy ◽  
Plant Cells ◽  
Preprophase Band ◽  
Guard Cells ◽  
Cell Types ◽  
Cell Cortex ◽  
Stomatal Complexes

Immunofluorescence microscopy has proven to be a valuable accompaniment to electron microscopy for study of the cytoskeleton of plant cells. Whereas electron microscopy provides greater resolution and details of the spatial relationships of the cytoskeleton to other cellular components, fluorescence visualization makes it possible to see the three-dimensional organization of cytoskeletal elements without laborious reconstruction of views from serial sections. An area in which immunofluorescence microscopy has been useful is the investigation of how plant cells organize and position the various microtubule arrays that are utilized during mitosis, cytokinesis and cell expansion phases. One of the earliest indications of an impending division in a meristematic plant cell is the formation of a preprophase band of microtubules in the cell cortex, at the site where the new wall will be placed at the subsequent cytokinesis. At its later stages, the band is narrower than when first identifiable. In most cells, preprophase band microtubules have the same general orientation as the preceding interphase microtubules, and so preprophase band formation here could, in theory, be achieved by lateral bundling of microtubules.Cells in which the division site and the preprophase band that marks it are not oriented parallel to interphase microtubules are found in stomatal complexes of grass leaves . Fig. 1 illustrates the arrangement of two such cell types: the guard mother cell, which divides lengthwise to form two guard cells, side-by-side, and the subsidiary mother cell, which undergoes a very asymmetric division to produce one of the pair of lens-shaped subsidiary cells that flank the guard cells. Interphase and preprophase arrangements of microtubules for each cell type are diagrammed in Figs. 2-4. In order to examine how these cell types achieve the reorientation of microtubules that is necessary to progress from interphase to preprophase, sheets of epidermis containing actively dividing stomatal complex cells were examined with immunofluorescence microscopy using antibodies to tubulin. Thin epidermal slices of leaves were fixed and glued down to a slide, whereupon cell walls were enzymatically weakened so that unwanted cell layers could be removed . Because waves of division pass along grass leaves, cells of the same type in a given file tend to be at similar stages, which facilitates deduction of the developmental pattern.

scholarly journals Genome-Scale Analysis of the Uses of the Escherichia coli Genome: Model-Driven Analysis of Heterogeneous Data Sets

2003 ◽  
Vol 185 (21) ◽  
pp. 6392-6399 ◽  
Author(s):  
Timothy E. Allen ◽  
Markus J. Herrgård ◽  
Mingzhu Liu ◽  
Yu Qiu ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
In Silico ◽  
Half Life ◽  
Data Sets ◽  
Expression Data ◽  
E Coli ◽  
Model Driven ◽  
Life Data ◽  
Genome Scale

ABSTRACT The recent availability of heterogeneous high-throughput data types has increased the need for scalable in silico methods with which to integrate data related to the processes of regulation, protein synthesis, and metabolism. A sequence-based framework for modeling transcription and translation in prokaryotes has been established and has been extended to study the expression state of the entire Escherichia coli genome. The resulting in silico analysis of the expression state highlighted three facets of gene expression in E. coli: (i) the metabolic resources required for genome expression and protein synthesis were found to be relatively invariant under the conditions tested; (ii) effective promoter strengths were estimated at the genome scale by using global mRNA abundance and half-life data, revealing genes subject to regulation under the experimental conditions tested; and (iii) large-scale genome location-dependent expression patterns with approximately 600-kb periodicity were detected in the E. coli genome based on the 49 expression data sets analyzed. These results support the notion that a structured model-driven analysis of expression data yields additional information that can be subjected to commonly used statistical analyses. The integration of heterogeneous genome-scale data (i.e., sequence, expression data, and mRNA half-life data) is readily achieved in the context of an in silico model.

scholarly journals WrbA from Escherichia coli and Archaeoglobus fulgidus Is an NAD(P)H:Quinone Oxidoreductase

2006 ◽  
Vol 188 (10) ◽  
pp. 3498-3506 ◽  
Author(s):  
Eric V. Patridge ◽  
James G. Ferry
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Cell Types ◽  
Archaeoglobus Fulgidus ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Optimum Growth Temperature ◽  
Type Iv ◽  
Domains Of Life

ABSTRACT WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the Km NADH was 14 ± 0.43 μM and the Km benzoquinone was 5.8 ± 0.12 μM. For A. fulgidus WrbA, the Km NADH was 19 ± 1.7 μM and the Km benzoquinone was 37 ± 3.6 μM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.

scholarly journals ThePseudomonas aeruginosaInitiation Factor IF-2 Is Responsible for Formylation-independent Protein Initiation inP. aeruginosa

2004 ◽  
Vol 279 (50) ◽  
pp. 52262-52269 ◽  
Author(s):  
Marta Steiner-Mosonyi ◽  
Carole Creuzenet ◽  
Robert A. B. Keates ◽  
Benjamin R. Strub ◽  
Dev Mangroo
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Positive Charge ◽  
Initiation Factor ◽  
E Coli ◽  
Charge Potential ◽  
Dependent Protein ◽  
Independent Protein

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily inEscherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that thePseudomonas aeruginosainitiation factor IF-2 is required for formylation-independent protein initiation inP. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. TheE. coliIF-2, which participates exclusively in formylation-dependent protein initiation inE. coli, was unable to facilitate utilization of Met-tRNAfMetin initiation inP. aeruginosa. However, theE. coliIF-2 was made to function in formylation-independent protein initiation inP. aeruginosaby decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in theP. aeruginosaIF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMetin protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMetin protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.

scholarly journals Protein Synthesis in Escherichia coli with Mischarged tRNA

2003 ◽  
Vol 185 (12) ◽  
pp. 3524-3526 ◽  
Author(s):  
Bokkee Min ◽  
Makoto Kitabatake ◽  
Carla Polycarpo ◽  
Joanne Pelaschier ◽  
Gregory Raczniak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Trna Synthetase ◽  
Wild Type ◽  
Wild Type Level ◽  
E Coli ◽  
Missense Mutant ◽  
Tryptophan Synthetase

ABSTRACT Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNAAsn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNAAsn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNAAsn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

scholarly journals Postsegregational Killing Mediated by the P1 Phage “Addiction Module” phd-doc Requires the Escherichia coli Programmed Cell Death SystemmazEF

2001 ◽  
Vol 183 (6) ◽  
pp. 2046-2050 ◽  
Author(s):  
Ronen Hazan ◽  
Boaz Sat ◽  
Myriam Reches ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Protein Synthesis ◽  
Programmed Cell Death ◽  
Killing Effect ◽  
E Coli ◽  
Lethal Action ◽  
Free Cells ◽  
Postsegregational Killing

ABSTRACT “Addiction modules” consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia colichromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated byphd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEFsystems.

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