scholarly journals In Vivo Transcription Kinetics of a Synthetic Gene Uninvolved in Stress-Response Pathways in Stressed Escherichia coli Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e109005 ◽  
Author(s):  
Anantha-Barathi Muthukrishnan ◽  
Antti Martikainen ◽  
Ramakanth Neeli-Venkata ◽  
Andre S. Ribeiro
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Synthetic Gene ◽  
Escherichia Coli Cells ◽  
Kinetics Of

scholarly journals Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

2010 ◽  
Vol 190 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Julio O. Ortiz ◽  
Florian Brandt ◽  
Valério R.F. Matias ◽  
Lau Sennels ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Spatial Organization ◽  
Three Dimensional ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Three Dimensional Configuration

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

scholarly journals Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cells

1991 ◽  
Vol 276 (3) ◽  
pp. 637-641 ◽  
Author(s):  
F F Craig ◽  
A C Simmonds ◽  
D Watmore ◽  
F McCapra ◽  
M R H White
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Intact Cells ◽  
Cos Cells ◽  
Escherichia Coli Cells ◽  
Luminescent Signal ◽  
The Difference ◽  
Kinetics Of

Five esters of luciferin were synthesized and compared with native luciferin as substrates for firefly luciferase expressed in live intact mammalian cells. The esters themselves were not substrates for purified luciferase, but four were substrates for a purified esterase and all appeared to be hydrolysed to luciferin within mammalian cells. At a substrate concentration of 0.01 mM, the peak luminescence from the cos cells expressing luciferase was up to 6-fold greater with the esters than with unmodified luciferin. At 0.1 mM, the difference between luciferin and the esters was decreased. The kinetics of the luminescent signal with the different luciferin esters varied significantly, indicating possible differences in the rates of uptake, breakdown and enzyme inhibition. The esters did not support luminescence from Escherichia coli cells expressing firefly luciferase, suggesting a lack of appropriate esterase activity in this particular strain. The esters could be useful for the assay of luciferase expression in intact mammalian cells when luciferin levels are limiting, for example in tissues, and in plants. Alternative luciferin derivatives may allow further improvements in sensitivity.

Kinetics of mercuric reduction in intact and permeabilized Escherichia coli cells

1990 ◽  
Vol 12 (11) ◽  
pp. 854-859 ◽  
Author(s):  
George P. Philippidis ◽  
Janet L. Schottel ◽  
Wei-Shou Hu
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli Cells ◽  
Kinetics Of

Alteration of adipocyte calcium homeostasis by Escherichia coli endotoxin

1985 ◽  
Vol 248 (3) ◽  
pp. R331-R338
Author(s):  
K. M. Nelson ◽  
J. A. Spitzer
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Calcium Homeostasis ◽  
Compartmental Analysis ◽  
In Vitro Exposure ◽  
Exchange Kinetics ◽  
Intracellular Binding ◽  
Kinetics Of

The present study evaluated calcium homeostasis in rat adipocytes after either in vivo or in vitro exposure to Escherichia coli endotoxin. Fat cells from endotoxin-treated rats showed an enhanced uptake of 45Ca. In an attempt to differentiate between 45Ca binding to the cell surface and intracellular 45Ca accumulation, adipocytes were exposed to 5 mM LaCl3. The amount of 45Ca remaining associated with lanthanum-treated adipocytes was taken to be located intracellularly and was increased in adipocytes from endotoxin-treated rats. The amount of 45Ca displaced by lanthanum was also increased in adipocytes from endotoxin-treated rats. This suggested that the endotoxin-induced increase of 45Ca accumulation included both cell surface and intracellular binding sites. Compartmental analysis of the exchange kinetics of cell-associated 45Ca with 40Ca in the medium indicated a 77% increase in the size of the cell surface compartment of adipocytes from endotoxin-treated rats compared with controls. In addition, endotoxin treatment altered the flux of calcium from the cells to the medium. In vitro exposure of freshly prepared adipocytes to 250 or 750 micrograms endotoxin/ml did not produce a perturbation of adipocyte calcium homeostasis. The results indicate that endotoxin induces alterations in the ability of adipocytes to regulate calcium translocations, suggesting that some metabolic and hormonal aspects of endotoxins' actions may be mediated through perturbation of cellular calcium homeostasis.

Kinetics of the bleomycin A2 damage in vivo to the folded chromosome of Escherichia coli

1983 ◽  
Vol 43 (2) ◽  
pp. 245-251 ◽  
Author(s):  
J.R. Pellon ◽  
A.J. Sinskey ◽  
S.M. Hecht ◽  
R.F. Gomez
Keyword(s):  
Escherichia Coli ◽  
Kinetics Of

scholarly journals Genome-wide comparison of phage M13-infected vs. uninfectedEscherichia coli

2005 ◽  
Vol 51 (1) ◽  
pp. 29-35 ◽  
Author(s):  
Fredrik Karlsson ◽  
Ann-Christin Malmborg-Hager ◽  
Ann-Sofie Albrekt ◽  
Carl A.K Borrebaeck
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Acid Stress ◽  
Phage Infection ◽  
Transcriptional Analysis ◽  
Filamentous Phage ◽  
Phosphotransferase System ◽  
E Coli ◽  
Genome Wide

To identify Escherichia coli genes potentially regulated by filamentous phage infection, we used oligonucleotide microarrays. Genome-wide comparison of phage M13-infected and uninfected E. coli, 2 and 20 min after infection, was performed. The analysis revealed altered transcription levels of 12 E. coli genes in response to phage infection, and the observed regulation of phage genes correlated with the known in vivo pattern of M13 mRNA species. Ten of the 12 host genes affected could be grouped into 3 different categories based on cellular function, suggesting a coordinated response. The significantly upregulated genes encode proteins involved in reactions of the energy-generating phosphotransferase system and transcription processing, which could be related to phage transcription. No genes belonging to any known E. coli stress response pathways were scored as upregulated. Furthermore, phage infection led to significant downregulation of transcripts of the bacterial genes gadA, gadB, hdeA, gadE, slp, and crl. These downregulated genes are normally part of the host stress response mechanisms that protect the bacterium during conditions of acid stress and stationary phase transition. The phage-infected cells demonstrated impaired function of the oxidative and the glutamate-dependent acid resistance systems. Thus, global transcriptional analysis and functional analysis revealed previously unknown host responses to filamentous phage infection.Key words: filamentous phage infection, global transcriptional analysis, AR, Escherichia coli.

scholarly journals Bacterial protein translocation requires only one copy of the SecY complex in vivo

2012 ◽  
Vol 198 (5) ◽  
pp. 881-893 ◽  
Author(s):  
Eunyong Park ◽  
Tom A. Rapoport
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Protein Translocation ◽  
Cross Linking ◽  
Bacterial Protein ◽  
Oligomeric State ◽  
E Coli ◽  
Cotranslational Translocation ◽  
Escherichia Coli Cells

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.

Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature?

2012 ◽  
Vol 40 (5) ◽  
pp. 950-954 ◽  
Author(s):  
Andres Binolfi ◽  
Francois-Xavier Theillet ◽  
Philipp Selenko
Keyword(s):  
Escherichia Coli ◽  
Protein Structure ◽  
Intracellular Protein ◽  
E Coli ◽  
Intrinsically Disordered ◽  
Escherichia Coli Cells ◽  
Rule Out ◽  
In Cells

The notion that human α-synuclein is an intrinsically disordered monomeric protein was recently challenged by a postulated α-helical tetramer as the physiologically relevant protein structure. The fact that this alleged conformation had evaded detection for so many years was primarily attributed to a widely used denaturation protocol to purify recombinant α-synuclein. In the present paper, we provide in-cell NMR evidence obtained directly in intact Escherichia coli cells that challenges a tetrameric conformation under native in vivo conditions. Although our data cannot rule out the existence of other intracellular protein states, especially in cells of higher organisms, they indicate clearly that inside E. coli α-synuclein is mostly monomeric and disordered.

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