scholarly journals Bacterial ageing in the absence of external stressors

2019 ◽  
Vol 374 (1786) ◽  
pp. 20180442 ◽  
Author(s):  
Urszula Łapińska ◽  
Georgina Glover ◽  
Pablo Capilla-Lasheras ◽  
Andrew J. Young ◽  
Stefano Pagliara
Keyword(s):  
Damage Accumulation ◽  
Doubling Time ◽  
Fluorescent Proteins ◽  
Parental Cell ◽  
Light Sources ◽  
Strong Light ◽  
E Coli ◽  
Glucose Accumulation ◽  
Asymmetric Partitioning ◽  
Progressive Accumulation

Evidence of ageing in the bacterium Escherichia coli was a landmark finding in senescence research, as it suggested that even organisms with morphologically symmetrical fission may have evolved strategies to permit damage accumulation. However, recent work has suggested that ageing is only detectable in this organism in the presence of extrinsic stressors, such as the fluorescent proteins and strong light sources typically used to excite them. Here we combine microfluidics with brightfield microscopy to provide evidence of ageing in E. coli in the absence of these stressors. We report (i) that the doubling time of the lineage of cells that consistently inherits the ‘maternal old pole’ progressively increases with successive rounds of cell division until it reaches an apparent asymptote, and (ii) that the parental cell divides asymmetrically, with the old pole daughter showing a longer doubling time and slower glucose accumulation than the new pole daughter. Notably, these patterns arise without the progressive accumulation or asymmetric partitioning of observable misfolded-protein aggregates, phenomena previously hypothesized to cause the ageing phenotype. Our findings suggest that ageing is part of the naturally occurring ecologically-relevant phenotype of this bacterium and highlight the importance of alternative mechanisms of damage accumulation in this context. This article is part of a discussion meeting issue ‘Single cell ecology’.

scholarly journals Conjugation of Carboxylated Graphene Quantum Dots with Cecropin P1 for Bacterial Biosensing Applications

2020 ◽  
Author(s):  
Jonathan Bruce ◽  
Jude Clapper
Keyword(s):  
Quantum Dots ◽  
Structural Modification ◽  
Light Emission ◽  
Limit Of Detection ◽  
Graphene Quantum Dots ◽  
Strong Light ◽  
E Coli ◽  
Cecropin P1 ◽  
Compact Field ◽  
Test Kit

<p>Quantum dots have proven to be strong candidates for biosensing applications in recent years, due to their strong light emission properties and their ability to be modified with a variety of functional groups for the detection of different analytes. Here, we investigate the use of conjugated carboxylated graphene quantum dots (CGQDs) for the detection of <i>E. coli</i>, using a biosensing procedure that focuses on measuring changes in fluorescence quenching. We have also further developed this biosensing assay into a compact, field-deployable test kit focused on rapidly measuring changes in absorbance to determine bacterial concentration. Our CGQDs were conjugated with cecropin P1, a naturally-produced antibacterial peptide that facilitates the attachment of CGQDs to <i>E. coli</i> cells. We also confirm the structural modification of these conjugated CGQDs in addition to analyzing their optical characteristics. Our findings have the potential to be used in situations where rapid, reliable detection of bacteria in liquids, such as drinking water, is required, especially given our biosensor’s relatively low observed limit of detection (LOD).</p><br>

Chemostat modeling ofEscherichia colipersistence in conventionalized mono-associated and streptomycin-treated mice

2001 ◽  
Vol 47 (1) ◽  
pp. 86-90 ◽  
Author(s):  
Camilla Rang ◽  
Tore Midtvedt ◽  
Søren Molin ◽  
Lin Chao
Keyword(s):  
Escherichia Coli ◽  
Doubling Time ◽  
Keystone Species ◽  
Intestinal Bacteria ◽  
Intestinal Colonization ◽  
Chemostat Model ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Intestinal Ecology

We have previously shown that Escherichia coli BJ4 has similar doubling time in mice that are mono-associated (having only the inoculated E. coli BJ4) or streptomycin-treated (having mainly gram-positive bacteria plus the inoculated E. coli BJ4). We also showed that when the mice were conventionalized (fed cecum homogenate from conventional mice or ones with a complete microbial flora), the introduction of complete flora in both cases increased the in vivo doubling time, while decreasing the colony counts in fecal samples. To determine whether the increase in doubling time could explain the decrease in colony counts, we analyzed our previous results by a chemostat model. The analysis shows that the increasing doubling time alone is sufficient to explain the decrease in colony counts in mono-associated mice, but not in the streptomycin-treated mice. The observed decreasing rate in colony counts in streptomycin-treated mice is slower than predicted. Furthermore, whereas the model predicted a decrease to extinction in both mice, the E. coli persist at a frequency 10-80 times higher in streptomycin-treated mice than in mono-associated mice. Thus, while a chemostat model is able to explain some of the population dynamics of intestinal bacteria in mice, additional factors not included in the model are stabilizing the system. Because we find that E. coli declines more slowly and to a higher stabilization frequency in streptomycin-treated mice, which have a more diverse flora before conventionalization, we take these results to suggest that the persistence of E. coli populations is promoted by species diversity. We propose that a mechanism for the persistence may be the presence of new E. coli niches created by keystone species in the more diverse flora.Key words: intestinal ecology, intestinal colonization, chemostat, keystone species, conventionalization.

scholarly journals Spatial engineering of E. coli with addressable phase-separated RNAs

2020 ◽  
Author(s):  
Haotian Guo ◽  
Joseph C. Ryan ◽  
Adeline Mallet ◽  
Xiaohu Song ◽  
Victor Pabst ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Fluorescent Proteins ◽  
Mrna Translation ◽  
Cag Repeats ◽  
Scaffolding Protein ◽  
Branch Points ◽  
Multilayered Structures ◽  
E Coli ◽  
Versatile Tool

AbstractBiochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within the Escherichia coli. TEARS are assembled from RNA-binding protein recruitment domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures and a non-equilibrium mechanism controlling their composition and reaction robustness. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. TEARS are a simple and versatile tool for spatially controlling E. coli biochemistry.

scholarly journals Coordination between E. coli Cell Size and Cell Cycle Mediated by DnaA

2020 ◽  
Author(s):  
Qing Zhang ◽  
Zhichao Zhang ◽  
Hualin Shi
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Dna Replication ◽  
Cell Size ◽  
Doubling Time ◽  
Cell Mass ◽  
Replication Initiation ◽  
General Relationship ◽  
E Coli ◽  
Regulatory Processes

Sixty years ago, bacterial cell size was found as an exponential function of growth rate. Fifty years ago, a more general relationship was proposed, in which the cell mass was equal to the initiation mass multiplied by the ratio of the total time of the C and D periods to the doubling time. This relationship has recently been experimentally confirmed by perturbing doubling time, C period, D period or the initiation mass. However, the underlying molecular mechanism remains unclear. Here, we developed a mechanistic and kinetic model to describe how the initiator protein DnaA mediates the initiation of DNA replication in E. coli. In the model, we introduced an initiation probability function involving competitive binding of DnaA-ATP (active) and DnaA-ADP (inactive) at replication origin to determine the initiation of replication. In addition, we considered RNAP availability, ppGpp inhibition, DnaA autorepression, DnaA titration by chromosomal sites, hydrolysis of DnaA-ATP along with DNA replication, reactivation of DnaA-ADP and established a kinetic description of these DnaA regulatory processes. We simulated DnaA kinetics and obtained a self-consistent cell size and a regular DnaA oscillation coordinated with the cell cycle at steady state. The relationship between the cell size obtained by the simulation and the growth rate, C period, D period or initiation mass reproduces the results of the experiment. This model also predicts how the number of DnaA and the initiation mass vary with the perturbation parameters (including those reflecting the mutation or interference of DnaA regulatory processes), which is comparable to experimental data. The results suggest that the regulatory mechanisms of DnaA level and activity are associated with the invariance of initiation mass and the cell size general relationship for matching frequencies of replication initiation and cell division. This study may provide clues for concerted control of cell size and cell cycle in synthetic biology.

scholarly journals A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

2020 ◽  
Author(s):  
Manabu Murakami ◽  
Agnieszka M. Murakami ◽  
Kazuyoshi Hirota ◽  
Shirou Itagaki
Keyword(s):  
Topoisomerase Ii ◽  
Fluorescent Proteins ◽  
Expression System ◽  
Simple Expression ◽  
Amino Acid Sequences ◽  
Expression Plasmid ◽  
Iptg Induction ◽  
E Coli ◽  
Mammalian Expression ◽  
Effective Selection

AbstractWe introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-β-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel β2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

scholarly journals Ceftriaxone Degradation by Titanium Dioxide (TiO2) Nanoparticles: Toxicity and Degradation Mechanism

2020 ◽  
Vol 6 (1) ◽  
pp. 82-89
Author(s):  
Mohammad Rofik Usman ◽  
Azmi Prasasti ◽  
Sovia Islamiah ◽  
Alfian Nur Firdaus ◽  
Ayu Wanda Marita ◽  
...  
Keyword(s):  
Titanium Dioxide ◽  
Tio2 Nanoparticles ◽  
Degradation Mechanism ◽  
Degradation Process ◽  
Light Sources ◽  
E Coli ◽  
Cephalosporin Antibiotics ◽  
Uv Visible ◽  
Uv Lamp ◽  
Degradation Degree

Ceftriaxone is a third generation of cephalosporin antibiotics that commonly used in patients with ulcers. Ceftriaxone residues in the environment are degraded using Titanium dioxide (TiO2) nanoparticles. Degradation of ceftriaxone using TiO2 nanoparticles was influenced by environmental conditions, such as light sources, pH of the solution, the mass of TiO2 nanoparticles, and the length of radiation. The remained ceftriaxone was analyzed by using a spectrophotometer UV-visible. The toxicity of the solution after the degradation process was tested on Escherichia coli and the type of products resulted was analyzed using Liquid Chromatography-Mass Spectrophotometry (LC-MS). The optimum conditions in degrading 50 mL 250 ppm ceftriaxone was radiation under a mercury UV lamp (white), pH 8, and 100 mg of TiO2 nanoparticles for 9 hours. The degradation degree of ceftriaxone obtained was 96.52%, producing simpler compounds that not toxic to E. Coli.

Fluorescence Microscopy Light Sources

2012 ◽  
Vol 20 (4) ◽  
pp. 22-28 ◽  
Author(s):  
Kavita Aswani ◽  
Tushare Jinadasa ◽  
Claire M. Brown
Keyword(s):  
Fluorescence Microscopy ◽  
Visible Light ◽  
White Light ◽  
Fluorescent Proteins ◽  
Life Sciences ◽  
Light Sources ◽  
Light Spectrum ◽  
Physical Sciences ◽  
Light Emitting ◽  
Microscopy Techniques

Fluorescence microscopy techniques are now prevalent throughout the life sciences and many of the physical sciences. These techniques are often dependent on white light sources that have evolved from the more traditional mercury arc lamp to metal halide sources to the more recent light emitting diodes (LEDs). The newer light sources show more uniform power across the visible light spectrum, allowing for the use of fluorophores and fluorescent proteins outside of the peak wavelengths associated with the more traditional light sources.

STEAM Connections: Painting with Bacteria

2018 ◽  
Vol 80 (4) ◽  
pp. 305-307
Author(s):  
Regina Wu ◽  
Caren Brinkema ◽  
Michaela Peterson ◽  
Adam Waltzer ◽  
Jeanne Chowning
Keyword(s):  
Gene Expression ◽  
Multidisciplinary Approach ◽  
Fluorescent Proteins ◽  
E Coli ◽  
Works Of Art ◽  
Different Types ◽  
Related Science ◽  
Science Activities

Arts-related science activities provide unique opportunities to engage students' strengths and motivate different types of learners (Jolly, 2014). Incorporating arts into the discussion of gene expression and microbiology introduces students to a multidisciplinary approach to STEM and provides an opportunity to explore the use of science in different fields such as design, art, and industry. In this protocol extension students create living works of art on agar plates by “painting” with E. coli that express fluorescent proteins of various colors.

scholarly journals Design of a biosensor for direct visualisation of auxin

2020 ◽  
Author(s):  
Ole Herud-Sikimic ◽  
Andre C. Stiel ◽  
Marina Ortega-Perez ◽  
Sooruban Shanmugaratnam ◽  
Birte Höcker ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Detection System ◽  
Temporal Distribution ◽  
Binding Pocket ◽  
Irreversible Processes ◽  
E Coli ◽  
Cellular Resolution ◽  
Plant Life ◽  
Transport Inhibitors

In plants, one of the most important regulative small molecules is indole-3-acetic acid (IAA) known as auxin. Its dynamic redistribution plays an essential role in virtually every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, the spatial and temporal distribution of auxin at cellular resolution could not be determined directly. Instead it has been inferred from visualisation of irreversible processes involving the endogenous auxin response machinery3-7. This detection system failed to record transient changes. Here we report on a genetically encoded biosensor for quantitative in vivo visualisation of auxin distributions. The sensor is based on the E. coli tryptophan repressor (TrpR)8 whose binding pocket was engineered for specific IAA binding and coupled to fluorescent proteins to employ FRET as readout. This sensor, unlike previous systems, enables direct monitoring of the fast uptake and clearance of auxin by individual cells in the plant as well as the graded spatial distribution along the root axis and its perturbation by transport inhibitors. Thus, our auxin sensor enables mapping of auxin concentrations at (sub)cellular resolution and their changes in time and space during plant life.

scholarly journals Synthetic lipid-containing scaffolds enhance production by co-localizing enzymes

2016 ◽  
Author(s):  
Cameron Myhrvold ◽  
Jessica K. Polka ◽  
Pamela A. Silver
Keyword(s):  
Building Materials ◽  
Fluorescent Proteins ◽  
Biological Activities ◽  
Live Cells ◽  
Enzymatic Production ◽  
E Coli ◽  
Subcellular Organization ◽  
Synthetic Scaffolds ◽  
Indigo Production ◽  
20 Nm

AbstractSubcellular organization is critical for isolating, concentrating, and protecting biological activities. Natural subcellular organization is often achieved using co-localization of proteins on scaffold molecules, thereby enhancing metabolic fluxes and enabling co-regulation. Synthetic scaffolds extend these benefits to new biological processes, and are typically constructed from proteins or nucleic acids. To expand the range of available building materials, we use a minimal set of components from the lipid-encapsulated bacteriophage Φ6 to form synthetic lipid-containing scaffolds (SLSs) inE. coli. Analysis of diffusive behavior by tracking particles in live cells indicates that SLSs are >20 nm in diameter; furthermore, density measurements demonstrate that SLSs contain a mixture of lipids and proteins. The fluorescent proteins mCitrine and mCerulean can be co-localized to SLSs. To test for effects on enzymatic production, we localized two enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent increase in indigo production, showing that SLSs can enhance metabolic reactions.

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