scholarly journals Engineering natural competence into the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

2021 ◽  
Author(s):  
Kristen E. Wendt ◽  
Patricia Walker ◽  
Annesha Sengupta ◽  
Justin Ungerer ◽  
Himadri B. Pakrasi
Keyword(s):  
Natural Transformation ◽  
Model Organism ◽  
Model System ◽  
Research Community ◽  
Synechococcus Elongatus ◽  
Model Systems ◽  
Extracellular Dna ◽  
Natural Competence ◽  
Fast Growing ◽  
Cyanobacterial Species

Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism’s inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA . By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain ( Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.

scholarly journals Mobilising molluscan models and genomes in biology

2020 ◽  
Author(s):  
Angus Davison ◽  
Maurine Neiman
Keyword(s):  
Cell Lines ◽  
Model System ◽  
Research Community ◽  
Model Systems ◽  
Current Application ◽  
Animal Evolution ◽  
Applied Model ◽  
Relative Neglect ◽  
Mollusc Species ◽  
Genome Assemblies

Molluscs are amongst the most ancient, diverse, and important of all animal taxa. Even so, no individual mollusc species has emerged as a broadly applied model system in biology. We here make the case that both perceptual and methodological barriers have played a role in the relative neglect of molluscs as research organisms. We then summarize the current application and potential of molluscs and their genomes to address important questions in animal biology, and the state of the field when it comes to the availability of resources such as genome assemblies, cell lines, and other key elements necessary to mobilising the development of molluscan model systems. We conclude by contending that a cohesive research community that works together to elevate multiple molluscan systems to ‘model’ status will create new opportunities in addressing basic and applied biological problems, including general features of animal evolution.

scholarly journals Mobilizing molluscan models and genomes in biology

2021 ◽  
Vol 376 (1825) ◽  
Author(s):  
Angus Davison ◽  
Maurine Neiman
Keyword(s):  
Model System ◽  
Research Community ◽  
Model Systems ◽  
Current Application ◽  
Future Directions ◽  
Animal Evolution ◽  
Applied Model ◽  
Relative Neglect ◽  
Mollusc Species ◽  
Genome Assemblies

Molluscs are among the most ancient, diverse, and important of all animal taxa. Even so, no individual mollusc species has emerged as a broadly applied model system in biology. We here make the case that both perceptual and methodological barriers have played a role in the relative neglect of molluscs as research organisms. We then summarize the current application and potential of molluscs and their genomes to address important questions in animal biology, and the state of the field when it comes to the availability of resources such as genome assemblies, cell lines, and other key elements necessary to mobilising the development of molluscan model systems. We conclude by contending that a cohesive research community that works together to elevate multiple molluscan systems to ‘model’ status will create new opportunities in addressing basic and applied biological problems, including general features of animal evolution. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

scholarly journals Singularly Perturbed Oscillators with Exponential Nonlinearities

2021 ◽  
Author(s):  
S. Jelbart ◽  
K. U. Kristiansen ◽  
P. Szmolyan ◽  
M. Wechselberger
Keyword(s):  
Limit Cycles ◽  
Space Dimension ◽  
Blow Up ◽  
Exponential Convergence ◽  
Model System ◽  
Piecewise Smooth ◽  
Singularly Perturbed ◽  
Model Systems ◽  
Smooth System ◽  
Unique Limit

AbstractSingular exponential nonlinearities of the form $$e^{h(x)\epsilon ^{-1}}$$ e h ( x ) ϵ - 1 with $$\epsilon >0$$ ϵ > 0 small occur in many different applications. These terms have essential singularities for $$\epsilon =0$$ ϵ = 0 leading to very different behaviour depending on the sign of h. In this paper, we consider two prototypical singularly perturbed oscillators with such exponential nonlinearities. We apply a suitable normalization for both systems such that the $$\epsilon \rightarrow 0$$ ϵ → 0 limit is a piecewise smooth system. The convergence to this nonsmooth system is exponential due to the nonlinearities we study. By working on the two model systems we use a blow-up approach to demonstrate that this exponential convergence can be harmless in some cases while in other scenarios it can lead to further degeneracies. For our second model system, we deal with such degeneracies due to exponentially small terms by extending the space dimension, following the approach in Kristiansen (Nonlinearity 30(5): 2138–2184, 2017), and prove—for both systems—existence of (unique) limit cycles by perturbing away from singular cycles having desirable hyperbolicity properties.

scholarly journals Paracoccus denitrificans: a genetically tractable model system for studying respiratory complex I

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Owen D. Jarman ◽  
Olivier Biner ◽  
John J. Wright ◽  
Judy Hirst
Keyword(s):  
Complex I ◽  
Paracoccus Denitrificans ◽  
Model System ◽  
Proton Pumping ◽  
Model Systems ◽  
Metabolic Enzyme ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Mitochondrial Complex I ◽  
Mitochondrial Complex ◽  
Inner Mitochondrial Membrane

AbstractMitochondrial complex I (NADH:ubiquinone oxidoreductase) is a crucial metabolic enzyme that couples the free energy released from NADH oxidation and ubiquinone reduction to the translocation of four protons across the inner mitochondrial membrane, creating the proton motive force for ATP synthesis. The mechanism by which the energy is captured, and the mechanism and pathways of proton pumping, remain elusive despite recent advances in structural knowledge. Progress has been limited by a lack of model systems able to combine functional and structural analyses with targeted mutagenic interrogation throughout the entire complex. Here, we develop and present the α-proteobacterium Paracoccus denitrificans as a suitable bacterial model system for mitochondrial complex I. First, we develop a robust purification protocol to isolate highly active complex I by introducing a His6-tag on the Nqo5 subunit. Then, we optimize the reconstitution of the enzyme into liposomes, demonstrating its proton pumping activity. Finally, we develop a strain of P. denitrificans that is amenable to complex I mutagenesis and create a catalytically inactive variant of the enzyme. Our model provides new opportunities to disentangle the mechanism of complex I by combining mutagenesis in every subunit with established interrogative biophysical measurements on both the soluble and membrane bound enzymes.

scholarly journals Behavioral Effects of a Chemorepellent Receptor Knockout Mutation in Tetrahymena thermophila

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Dianxiong Zou ◽  
Todd M. Hennessey
Keyword(s):  
Receptor Gene ◽  
Classical Model ◽  
Model System ◽  
Model Systems ◽  
Unicellular Eukaryote ◽  
Wild Type ◽  
Inhibitory Neurotransmitter ◽  
In The Wild ◽  
Sensory Responses ◽  
Major Emphasis

ABSTRACT Although many single-cell eukaryotes have served as classical model systems for chemosensory studies for decades, the major emphasis has been on chemoattraction and no chemorepellent receptor gene has been identified in any unicellular eukaryote. This is the first description of a gene that codes for a chemorepellent receptor in any protozoan. Integration of both depolarizing chemorepellent pathways and hyperpolarizing chemoattractant pathways is as important to chemoresponses of motile unicells as excitatory and inhibitory neurotransmitter pathways are to neurons. Therefore, both chemoattractant and chemorepellent pathways should be represented in a useful unicellular model system. Tetrahymena cells provide such a model system because simple behavioral bioassays, gene knockouts, biochemical analysis, and other approaches can be used with these eukaryotic model cells. This work can contribute to the basic understanding of unicellular sensory responses and provide insights into the evolution of chemoreceptors and possible chemorepellent approaches for preventing infections by some pathogenic protozoa. A conditioned supernatant from Tetrahymena thermophila contains a powerful chemorepellent for wild-type cells, and a gene called G37 is required for this response. This is the first genomic identification of a chemorepellent receptor in any eukaryotic unicellular organism. This conditioned supernatant factor (CSF) is small (<1 kDa), and its repellent effect is resistant to boiling, protease treatment, and nuclease digestion. External BAPTA eliminated the CSF response, suggesting that Ca2+ entry is required for the classical avoiding reactions (AR) used for chemorepulsion. A macronuclear G37 gene knockout (G37-KO) mutant is both nonresponsive to the CSF and overresponsive to other repellents such as quinine, lysozyme, GTP, and high potassium concentrations. All of these mutant phenotypes were reversed by overexpression of the wild-type G37 gene in a G37 overexpression mutant. Overexpression of G37 in the wild type caused increased responsiveness to the CSF and underresponsiveness to high K+ concentrations. Behavioral adaptation (by prolonged exposure to the CSF) caused decreases in responsiveness to all of the stimuli used in the wild type and the overexpression mutant but not in the G37-KO mutant. We propose that the constant presence of the CSF causes a decreased basal excitability of the wild type due to chemosensory adaptation through G37 and that all of the G37-KO phenotypes are due to an inability to detect the CSF. Therefore, the G37 protein may be the CSF receptor. The physiological role of these G37-mediated responses may be to both moderate basal excitability and detect the CSF as an indicator of high cell density growth. IMPORTANCE Although many single-cell eukaryotes have served as classical model systems for chemosensory studies for decades, the major emphasis has been on chemoattraction and no chemorepellent receptor gene has been identified in any unicellular eukaryote. This is the first description of a gene that codes for a chemorepellent receptor in any protozoan. Integration of both depolarizing chemorepellent pathways and hyperpolarizing chemoattractant pathways is as important to chemoresponses of motile unicells as excitatory and inhibitory neurotransmitter pathways are to neurons. Therefore, both chemoattractant and chemorepellent pathways should be represented in a useful unicellular model system. Tetrahymena cells provide such a model system because simple behavioral bioassays, gene knockouts, biochemical analysis, and other approaches can be used with these eukaryotic model cells. This work can contribute to the basic understanding of unicellular sensory responses and provide insights into the evolution of chemoreceptors and possible chemorepellent approaches for preventing infections by some pathogenic protozoa.

Effect of Oxidized Myofibrils Protein Subjected to Mutiple Freeze-Thaw Cycles on N-Nitrosamine Formation in In Vitro Model System

2012 ◽  
Vol 550-553 ◽  
pp. 1590-1594 ◽  
Author(s):  
Hua Yang ◽  
Pei Pei Meng ◽  
Rui Wang ◽  
Pei Ran Li ◽  
Peng Li ◽  
...  
Keyword(s):  
Sodium Nitrite ◽  
Protein Carbonyl ◽  
Model System ◽  
Model Systems ◽  
Secondary Amines ◽  
Freeze Thaw ◽  
Heavy Chains ◽  
Carcinogenic Substance ◽  
The Times

N-nitrosamine is a kind of carcinogenic substance, which is possibly formed in the reaction of nitrites with amino acids or secondary amines. Two in vitro model systems were designed to evaluate the influence of oxidized myofibrils protein subjected to repeated freeze-thaw cycles (0, 1, 2, 3, 4, 7, 10 times) on N-nitrosamine formation. Model system I contains diethylamine and sodium nitrite, while model system II contains only sodium nitrite as reaction solution. Oxidized myofibrils protein were added to both systems. The results revealed that as the number of freeze-thaw cycles increased, cross-linking of myosin heavy chains and the content of protein carbonyl increased, but the content of protein sulfydryl decreased, which indicates oxidization of protein occurred. The concentration of N-nitrosodiethylamine increased as the number of freeze-thaw cycles increased, especially after four cycles. Oxidized myofibrils protein promoted the formation of N-nitrosodiethylamine. The more the times of freeze-thaw cycles were subjected, the more oxidization of myofibrils protein occurred and the higher yield of the N-nitrosodiethylamine.

scholarly journals Serum Albumin and Ca2+Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii

2016 ◽  
Vol 60 (8) ◽  
pp. 4920-4929 ◽  
Author(s):  
German Matias Traglia ◽  
Brettni Quinn ◽  
Sareda T. J. Schramm ◽  
Alfonso Soler-Bistue ◽  
Maria Soledad Ramirez
Keyword(s):  
Acinetobacter Baumannii ◽  
Natural Transformation ◽  
Hospital Environment ◽  
Human Pathogen ◽  
Nosocomial Pathogen ◽  
Natural Competence ◽  
Exogenous Dna ◽  
Content Type ◽  
Resistance Determinants ◽  
Main Protein

ABSTRACTThe increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition.Acinetobacter baumanniiis a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence inA. baumanniiare unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca2+or albumin. We show thatcomEAandpilQare involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence inA. baumannii. Overall, our results suggest that the main protein in blood enhances HGT inA. baumannii, contributing to the increase of AMR in this threatening human pathogen.

scholarly journals THE STUDY OF LOW-LACTOSE MILK WHEY STRUCTURE AND MODEL SYSTEMS ON ITS BASIS

2020 ◽  
Vol 3 ◽  
pp. 38-48
Author(s):  
Victoriya Gnitsevych ◽  
Tatiana Yudina ◽  
Yuliia Honchar ◽  
Olena Vasylieva ◽  
Liudmyla Diachuk
Keyword(s):  
Particle Size ◽  
Fractional Composition ◽  
Raw Materials ◽  
Model System ◽  
Model Systems ◽  
Equivalent Diameter ◽  
Contact Method ◽  
Technological Properties ◽  
Milk Whey ◽  
Crystal Diameter

This study developed a technology of low-lactose semi-finished products, based on fermented whey and pumpkin pulp puree, and offered a possibility of its use in the technology of structured culinary products. This research carried out the required substantiation of the methods of preliminary processing of raw materials, and studied the technological properties and structure of model compositions with their use. During the experiment, a number of studies were carried out, which substantiated the method and modes of condensation of whey, and provided a comparative analysis of the homogeneity of lactose-free and lactose-containing samples of whey under various modes of condensation. The study obtained the results of calculations of the equivalent diameter of the studied samples of lactose-containing and low-lactose whey, condensed by the contact method and in vacuum. It was found, that the structure is homogeneous at a number average crystal diameter of up to 5 μm. The restriction is valid for CLLWV with a calculated diameter of about 3.84 μm with a coefficient of variation of 1.35 % with an increase of 10,000 times. The study revealed the alternation of smooth and granular sections of the micron level (0.1 ... 5 μm) in the structure of the studied low-lactose semi-finished product with an increase of 300 times. It was determined, that the extremum of the differential curve of the particle size distribution of CLLWV corresponds to the number average crystal diameter of 3.84 μm. It was established, that the most homogeneous fractional composition is inherent in the studied sample of CLLWV, for which the values of fraction diameters are in the range from 1.46 μm to 4.96 μm. The optimal ratio of the components of the model CLLWV: FPPP system was determined as 70 % to 30 % respectively. With this composition, the model system is characterized by the formation of protein-pectin complexes, which is confirmed by microscopy with a magnification of 90 times

scholarly journals CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

2021 ◽  
Vol 12 ◽  
Author(s):  
Edith Uetz-von Allmen ◽  
Guerric P. B. Samson ◽  
Vladimir Purvanov ◽  
Takahiro Maeda ◽  
Daniel F. Legler
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Neoplastic Cell ◽  
Model System ◽  
Model Systems ◽  
Cellular Model ◽  
Gm Csf ◽  
Endogenous Expression ◽  
Dendritic Cell Migration ◽  
Spatio Temporal

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

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