scholarly journals Using a Riboswitch Sensor to Detect Co2+/Ni2+ Transport in E. coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaoying Wang ◽  
Wei Wei ◽  
Jing Zhao
Keyword(s):  
Fluorescent Protein ◽  
Whole Cell ◽  
E Coli ◽  
Detection Process ◽  
Cellular Processes ◽  
Highly Sensitive ◽  
Resistance System ◽  
Intracellular Concentrations ◽  
In Cells

Intracellular concentrations of essential mental ions must be tightly maintained to avoid metal deprivation and toxicity. However, their levels in cells are still difficult to monitor. In this report, the combination of a Co2+Ni2+-specific riboswitch and an engineered downstream mCherry fluorescent protein allowed a highly sensitive and selective whole-cell Co2+/Ni2+ detection process. The sensors were applied to examine the resistance system of Co2+/Ni2+in E. coli, and the sensors were able to monitor the effects of genetic deletions. These results indicate that riboswitch-based sensors can be employed in the study of related cellular processes.

scholarly journals Development of an Autofluorescent Whole-Cell Biocatalyst by Displaying Dual Functional Moieties on Escherichia coli Cell Surfaces and Construction of a Coculture with Organophosphate-Mineralizing Activity

2008 ◽  
Vol 74 (24) ◽  
pp. 7733-7739 ◽  
Author(s):  
Chao Yang ◽  
Yaran Zhu ◽  
Jijian Yang ◽  
Zheng Liu ◽  
Chuanling Qiao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Ice Nucleation Protein ◽  
Whole Cell ◽  
E Coli ◽  
Dual Functional ◽  
Whole Cell Biocatalyst ◽  
Mineralizing Activity ◽  
Green Fluorescent

ABSTRACT Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.

scholarly journals Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging

2020 ◽  
Vol 162 ◽  
pp. 112258 ◽  
Author(s):  
Negin Zaraee ◽  
Fulya Ekiz kanik ◽  
Abdul Muyeed Bhuiya ◽  
Emily S. Gong ◽  
Matthew T. Geib ◽  
...  
Keyword(s):  
Label Free ◽  
Whole Cell ◽  
E Coli ◽  
Highly Sensitive ◽  
Digital Detection

scholarly journals Cell Division Defects in Escherichia coli Deficient in the Multidrug Efflux Transporter AcrEF-TolC

2005 ◽  
Vol 187 (22) ◽  
pp. 7815-7825 ◽  
Author(s):  
Sze Yi Lau ◽  
Helen I. Zgurskaya
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
E Coli ◽  
Membrane Fusion Protein ◽  
Green Fluorescent ◽  
Multiple Drugs ◽  
In Cells

ABSTRACT The Escherichia coli chromosome contains several operons encoding confirmed and predicted multidrug transporters. Among these transporters only the inactivation of components of the AcrAB-TolC complex leads to substantial changes in susceptibility to multiple drugs. This observation prompted a conclusion that other transporters are silent or expressed at levels insufficient to contribute to multidrug resistance phenotype. We found that increased expression of AcrA, the periplasmic membrane fusion protein, is toxic only in cells lacking the multidrug efflux transporter AcrEF. AcrEF-deficient cells with increased expression of AcrA have a severe cell division defect that results in cell filamentation (>50 μm). Similar defects were obtained in cells lacking the outer membrane channel TolC, which acts with AcrEF, suggesting that cell filamentation is caused by the loss of AcrEF function. Green fluorescent protein-AcrA fusion studies showed that in normal and filamentous cells AcrA is associated with membranes in a confined manner and that this localization is not affected by the lack of AcrEF. Similarly, the structure and composition of membranes were normal in filamentous cells. Fluorescence microscopy showed that the filamentous AcrEF-deficient E. coli cells are defective in chromosome condensation and segregation. Our results suggest that the E. coli AcrEF transporter is expressed under standard laboratory conditions and plays an important role in the normal maintenance of cell division.

scholarly journals Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

2009 ◽  
Vol 29 (13) ◽  
pp. 3665-3674 ◽  
Author(s):  
Alaattin Kaya ◽  
Huseyin C. Karakaya ◽  
Dmitri E. Fomenko ◽  
Vadim N. Gladyshev ◽  
Ahmet Koc
Keyword(s):  
Fluorescent Protein ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Boron Tolerance ◽  
Cellular Processes ◽  
Dna Library ◽  
Highly Sensitive ◽  
Genomic Dna Library ◽  
Major Facilitator ◽  
Green Fluorescent

ABSTRACT Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

scholarly journals Interphotoreceptor coupling: an evolutionary perspective

2021 ◽  
Author(s):  
Lorenzo Cangiano ◽  
Sabrina Asteriti
Keyword(s):  
Visual Information ◽  
Signal To Noise Ratio ◽  
Electrical Coupling ◽  
Physiological Role ◽  
Cellular Processes ◽  
Contact Points ◽  
Highly Sensitive ◽  
The Many ◽  
The Impact

AbstractIn the vertebrate retina, signals generated by cones of different spectral preference and by highly sensitive rod photoreceptors interact at various levels to extract salient visual information. The first opportunity for such interaction is offered by electrical coupling of the photoreceptors themselves, which is mediated by gap junctions located at the contact points of specialised cellular processes: synaptic terminals, telodendria and radial fins. Here, we examine the evolutionary pressures for and against interphotoreceptor coupling, which are likely to have shaped how coupling is deployed in different species. The impact of coupling on signal to noise ratio, spatial acuity, contrast sensitivity, absolute and increment threshold, retinal signal flow and colour discrimination is discussed while emphasising available data from a variety of vertebrate models spanning from lampreys to primates. We highlight the many gaps in our knowledge, persisting discrepancies in the literature, as well as some major unanswered questions on the actual extent and physiological role of cone-cone, rod-cone and rod-rod communication. Lastly, we point toward limited but intriguing evidence suggestive of the ancestral form of coupling among ciliary photoreceptors.

scholarly journals A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

2021 ◽  
Vol 22 (11) ◽  
pp. 6148
Author(s):  
Matteo Miceli ◽  
Silvana Casati ◽  
Pietro Allevi ◽  
Silvia Berra ◽  
Roberta Ottria ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Kinetic Constants ◽  
New Method ◽  
Monoacylglycerol Lipase ◽  
Enzymatic Cleavage ◽  
Highly Sensitive ◽  
Bioluminescence Assay ◽  
Identification And Characterization ◽  
In Cells

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

scholarly journals Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7

2019 ◽  
Vol 1081 ◽  
pp. 146-156 ◽  
Author(s):  
Susana Díaz-Amaya ◽  
Li-Kai Lin ◽  
Amanda J. Deering ◽  
Lia A. Stanciu
Keyword(s):  
Whole Cell ◽  
E Coli ◽  
Analytical Detection

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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