Faculty Opinions recommendation of Independent positioning and action of Escherichia coli replisomes in live cells.

2008 ◽  
Author(s):  
Nick Rhind
Keyword(s):  
Escherichia Coli ◽  
Live Cells

Differentiation of Viable and Dead Escherichia coli O157:H7 Cells on and in Apple Structures and Tissues following Chlorine Treatment

2002 ◽  
Vol 65 (2) ◽  
pp. 251-259 ◽  
Author(s):  
SCOTT L. BURNETT ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Live Cells ◽  
Confocal Scanning Laser Microscopy ◽  
Escherichia Coli O157 ◽  
Tissue Samples ◽  
E Coli ◽  
Sytox Green ◽  
Viable Cells ◽  
Nucleic Acid Stain ◽  
Protective Structures

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25°C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4°C, followed by treatment in water or chlorine solution at 21°C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers, as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodged in protective structures on the surface and within tissues of apples.

scholarly journals Independent Positioning and Action of Escherichia coli Replisomes in Live Cells

Cell ◽  
2008 ◽  
Vol 133 (1) ◽  
pp. 90-102 ◽  
Author(s):  
Rodrigo Reyes-Lamothe ◽  
Christophe Possoz ◽  
Olessia Danilova ◽  
David J. Sherratt
Keyword(s):  
Escherichia Coli ◽  
Live Cells

scholarly journals RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity

2021 ◽  
Author(s):  
Alex L. Payne-Dwyer ◽  
Aisha H. Syeda ◽  
Jack W. Shepherd ◽  
Lewis Frame ◽  
Mark. C. Leake
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Dna Repair ◽  
Mitomycin C ◽  
Single Molecule ◽  
Reca Protein ◽  
Live Cells ◽  
Dna Breaks ◽  
Fluorescent Reporters ◽  
Double Stranded Dna

AbstractThe RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA, RecBCD a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks and also facilitating decoration of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA and the RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting changes in stoichiometry, copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA filamental stoichiometries equivalent to several hundred molecules that act largely in RecA tetramers before DNA damage, but switch to approximately hexameric subunits when mature filaments are formed. Unexpectedly, we find that the physiologically predominant form of RecB is a dimer.

scholarly journals Recombinant Escherichia coli as a biofactory for various single- and multi-element nanomaterials

2018 ◽  
Vol 115 (23) ◽  
pp. 5944-5949 ◽  
Author(s):  
Yoojin Choi ◽  
Tae Jung Park ◽  
Doh C. Lee ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Chemical Species ◽  
Cell Extract ◽  
Live Cells ◽  
Biological Synthesis ◽  
Stable Chemical ◽  
Initial Ph

Nanomaterials (NMs) are mostly synthesized by chemical and physical methods, but biological synthesis is also receiving great attention. However, the mechanisms for biological producibility of NMs, crystalline versus amorphous, are not yet understood. Here we report biosynthesis of 60 different NMs by employing a recombinant Escherichia coli strain coexpressing metallothionein, a metal-binding protein, and phytochelatin synthase that synthesizes a metal-binding peptide phytochelatin. Both an in vivo method employing live cells and an in vitro method employing the cell extract are used to synthesize NMs. The periodic table is scanned to select 35 suitable elements, followed by biosynthesis of their NMs. Nine crystalline single-elements of Mn3O4, Fe3O4, Cu2O, Mo, Ag, In(OH)3, SnO2, Te, and Au are synthesized, while the other 16 elements result in biosynthesis of amorphous NMs or no NM synthesis. Producibility and crystallinity of the NMs are analyzed using a Pourbaix diagram that predicts the stable chemical species of each element for NM biosynthesis by varying reduction potential and pH. Based on the analyses, the initial pH of reactions is changed from 6.5 to 7.5, resulting in biosynthesis of various crystalline NMs of those previously amorphous or not-synthesized ones. This strategy is extended to biosynthesize multi-element NMs including CoFe2O4, NiFe2O4, ZnMn2O4, ZnFe2O4, Ag2S, Ag2TeO3, Ag2WO4, Hg3TeO6, PbMoO4, PbWO4, and Pb5(VO4)3OH NMs. The strategy described here allows biosynthesis of NMs with various properties, providing a platform for manufacturing various NMs in an environmentally friendly manner.

scholarly journals Increased Levels of Intracellular Calcium Are Not Required for the Formation of Attaching and Effacing Lesions by Enteropathogenic and Enterohemorrhagic Escherichia coli

1998 ◽  
Vol 66 (8) ◽  
pp. 3900-3908 ◽  
Author(s):  
Christopher Bain ◽  
Rogeria Keller ◽  
Georgina K. Collington ◽  
Luiz R. Trabulsi ◽  
Stuart Knutton
Keyword(s):  
Escherichia Coli ◽  
Intracellular Calcium ◽  
Bacterial Adhesion ◽  
Enterohemorrhagic Escherichia Coli ◽  
Live Cells ◽  
Free Calcium ◽  
Lesion Formation ◽  
E Coli ◽  
Infected Cells ◽  
Temporal And Spatial

ABSTRACT Elevated concentrations of intracellular calcium ([Ca]i) have been implicated as an important signalling event during attaching and effacing (A/E) lesion formation by enteropathogenic Escherichia coli (EPEC). The highly localized nature of the cytoskeletal and cell surface alterations occurring during A/E lesion formation suggests that there should be equally localized EPEC-induced signalling events. To analyze further the calcium responses to infection of HEp-2 cells by EPEC, we employed calcium-imaging fluorescence microscopy, which allows both temporal and spatial measurements of [Ca]i in live cells. Using this imaging technique, not only were we unable to detect any significant elevation in [Ca]i at sites of A/E EPEC adhesion, but, with several different classical EPEC and enterohemorrhagic E. coli (EHEC) strains and three different infection procedures, each of which resulted in extensive A/E bacterial adhesion, we were unable to detect any significant alterations in [Ca]i in infected cells compared to uninfected cells. In addition, chelation of intracellular free calcium with bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) did not, as previously reported, prevent A/E lesion formation. We conclude that increased [Ca]iare not required for A/E lesion formation by EPEC and EHEC.

scholarly journals Dead bacterial absorption of antimicrobial peptides underlies collective tolerance

2019 ◽  
Vol 16 (151) ◽  
pp. 20180701 ◽  
Author(s):  
Fan Wu ◽  
Cheemeng Tan
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptides ◽  
Mathematical Modelling ◽  
Dynamic Process ◽  
Bacterial Strain ◽  
Live Cells ◽  
Bacterial Cells ◽  
Tolerance Mechanisms ◽  
Cytoplasmic Membranes ◽  
Live Bacterial Cells

The collective tolerance towards antimicrobial peptides (APs) is thought to occur primarily through mechanisms associated with live bacterial cells. In contrast to the focus on live cells, we discover that the LL37 antimicrobial peptide kills a subpopulation of Escherichia coli , forming dead cells that absorb the remaining LL37 from the environment. Combining mathematical modelling with population and single-cell experiments, we show that bacteria absorb LL37 at a timing that coincides with the permeabilization of their cytoplasmic membranes. Furthermore, we show that one bacterial strain can absorb LL37 and protect another strain from killing by LL37. Finally, we demonstrate that the absorption of LL37 by dead bacteria can be reduced using a peptide adjuvant. In contrast to the known collective tolerance mechanisms, we show that the absorption of APs by dead bacteria is a dynamic process that leads to emergent population behaviour.

scholarly journals Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant

2011 ◽  
Vol 77 (17) ◽  
pp. 6133-6140 ◽  
Author(s):  
Yongjin Zhou ◽  
Lei Wang ◽  
Fan Yang ◽  
Xinping Lin ◽  
Sufang Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Essential Gene ◽  
Auxotrophic Mutant ◽  
Genetically Engineered ◽  
Reduced Form ◽  
Live Cells ◽  
Regulation Mechanism ◽  
Content Type ◽  
High Concentrations

ABSTRACTNAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels inEscherichia colicells that were genetically engineered to be NAD+auxotrophic. First, we expressed thentt4gene encoding the NAD(H) transporter in theE. colimutant YJE001, which had a deletion of thenadCgene responsible for NAD+de novobiosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential genenadE, which is responsible for the last step of NAD+biosynthesis in cells harboring thentt4gene. The minimal NAD+level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.

scholarly journals Intracellular absorption underlies collective bacterial tolerance towards an antimicrobial peptide

2018 ◽  
Author(s):  
Fan Wu ◽  
Cheemeng Tan
Keyword(s):  
Mathematical Modeling ◽  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Live Cells ◽  
Bacterial Cells ◽  
Tolerance Mechanisms ◽  
Intracellular Space ◽  
Cytoplasmic Membranes ◽  
New Directions ◽  
Live Bacterial Cells

AbstractThe collective tolerance towards antimicrobial peptides (APs) is thought to occur primarily through mechanisms associated with live bacterial cells. In contrast to the focus on live cells, we discover that the LL37 antimicrobial peptide kills Escherichia coli, forming a subpopulation of dead cells that absorbs the remaining LL37 into its intracellular space. Combining mathematical modeling with population and single-cell experiments, we show that bacteria absorb LL37 at a timing that coincides with the permeabilization of their cytoplasmic membranes. Furthermore, we show that one bacterial strain can absorb LL37 and protect another strain from killing by LL37. Finally, we demonstrate that the intracellular absorption of LL37 can be reduced using a peptide adjuvant. In contrast to the existing collective tolerance mechanisms, we show that the dead-bacterial absorption of APs is a dynamic process that leads to emergent population behavior, and the work suggests new directions to enhance the efficacy of APs.

scholarly journals Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification

2011 ◽  
Vol 77 (13) ◽  
pp. 4329-4335 ◽  
Author(s):  
Bilgin Taskin ◽  
Ayse Gul Gozen ◽  
Metin Duran
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Membrane Integrity ◽  
Dna Amplification ◽  
Live Cells ◽  
Dead Cell ◽  
Target Sequence ◽  
Propidium Monoazide ◽  
Indicator Organisms ◽  
Content Type

ABSTRACTQuantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed withEscherichia coliATCC 25922 as the model organism and theuidAgene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1.

scholarly journals Effects of injured and dead cells of Escherichia coli on the colony‐forming rate of live cells

FEBS Open Bio ◽  
2020 ◽  
Author(s):  
Mikako Saito ◽  
Norimasa Takatani ◽  
Tomonori Yoshida ◽  
Alvin Mariogani ◽  
Eol Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Live Cells
Sign in / Sign up

Export Citation Format

Share Document