scholarly journals A Simplified Protocol for Reversing Phenotypic Conversion of Ralstonia solanacearum during Experimentation

2020 ◽  
Vol 17 (12) ◽  
pp. 4274
Author(s):  
Pramod Kumar Sahu ◽  
Shailendra Singh ◽  
Amrita Gupta ◽  
Udai B. Singh ◽  
Surinder Paul ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Fluorescent Protein ◽  
Synergistic Effects ◽  
Dead Cell ◽  
Vascular Bundles ◽  
Virulent Type ◽  
Confocal Scanning Laser ◽  
The Stability ◽  
Green Fluorescent ◽  
Cell Fractions

Background: Ralstonia solanacearum has the problem of losing the virulence in laboratory conditions, during prolonged experimentation. Since pure colonies of R. solanacearum contain cell fractions differing in virulence, it was considered worthwhile to find a way of selecting the cells with lower attenuation. Therefore, a methodology for inducing virulent-type colonies occurrence in Ralstonia solanacearum was developed. Methods: Nutrient gradient was created by swabbing R. solanacearum culture in a slanted KMTTC medium, and Phyllanthus emblica extract was given by well diffusion. Live–dead cell imaging using BacLight, effects of ascorbic acid on cell viability, and production of virulence factors (exopolysaccharides, cellulase, and pectinase) supported this hypothesis. The tagging of R. solanacearum with green fluorescent protein and further confocal scanning laser microscopic visualization confirmed the colonization in vascular bundles of tomato. Results: P. emblica extract suppressed R. solanacearum initially in well diffusion, but further developed virulent-type colonies around the wells. Nutrient deprivation was found to have synergistic effects with P. emblica extract. The converted fluidal (virulent type) colonies could be able to colonize vascular bundles and cause wilting symptoms. Conclusion: This method will be useful in the laboratories working on biocontrol of R. solanacearum for maintaining virulent-type colonies. Moreover, it could form the basis for studies on the stability of phenotypic conversion and cell fractions in R. solanacearum.

Nanoparticles Composed by Oligochitosan and Polyethylenimine for Gene Delivery

2013 ◽  
Vol 284-287 ◽  
pp. 418-422
Author(s):  
Chi Hsien Liu ◽  
Mei Shan Cheng
Keyword(s):  
Gene Delivery ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Synergistic Effects ◽  
Image Analyzer ◽  
Transfection Efficacy ◽  
Fluorescence Protein ◽  
Green Fluorescent ◽  
3T3 Cell Lines

Cationic carriers including polyethylenimine, liposomes, and chitosan have been used to transfer plasmid DNA in vitro by condensing anionic DNA. Here, oligochitosan (OC) was found to have capacity for in-vitro gene delivery in four cell lines. Plasmid containing green fluorescent protein (GFP) gene was used as a reporter gene. The transfection efficacy and cell viability of the transfection vehicles were analyzed by using a high-throughput image analyzer. We found that DNA polyplexes formed by high dosage of OC could be efficiently delivered into the cells. The combination of OC and polyethylenimine (PEI) were found to significantly enhance the fluorescence protein expression. The introduction of oligochitosan in PEI-mediated transfection could increase the transfection efficacy and could reduce the toxicity of PEI. Additionally, the synergistic effects of PEI and OC were confirmed in CHO, Caco2, Hep-SK, and 3T3 cell lines. The detailed mechanism of PEI and oligochitosan on transfection was investigated by using gel retardation and DNase degradation experiments. A facile and inexpensive construction of gene delivering vehicles was developed herein by using oligochitosan and PEI.

scholarly journals Development and Dynamics of Pseudomonassp. Biofilms

2000 ◽  
Vol 182 (22) ◽  
pp. 6482-6489 ◽  
Author(s):  
Tim Tolker-Nielsen ◽  
Ulla C. Brinch ◽  
Paula C. Ragas ◽  
Jens Bo Andersen ◽  
Carsten Suhr Jacobsen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Physiological State ◽  
Cell Aggregation ◽  
Flow Chamber ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Scanning ◽  
Biofilm Bacteria ◽  
Fluorescent Cells ◽  
Confocal Scanning Laser ◽  
Green Fluorescent

ABSTRACT Pseudomonas sp. strain B13 and Pseudomonas putida OUS82 were genetically tagged with the green fluorescent protein and the Discosoma sp. red fluorescent protein, and the development and dynamics occurring in flow chamber-grown two-colored monospecies or mixed-species biofilms were investigated by the use of confocal scanning laser microscopy. Separate red or green fluorescent microcolonies were formed initially, suggesting that the initial small microcolonies were formed simply by growth of substratum attached cells and not by cell aggregation. Red fluorescent microcolonies containing a few green fluorescent cells and green fluorescent microcolonies containing a few red fluorescent cells were frequently observed in both monospecies and two-species biofilms, suggesting that the bacteria moved between the microcolonies. Rapid movement of P. putida OUS82 bacteria inside microcolonies was observed before a transition from compact microcolonies to loose irregularly shaped protruding structures occurred. Experiments involving a nonflagellated P. putida OUS82 mutant suggested that the movements between and inside microcolonies were flagellum driven. The results are discussed in relation to the prevailing hypothesis that biofilm bacteria are in a physiological state different from planktonic bacteria.

scholarly journals Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

2015 ◽  
Author(s):  
Anton Khmelinskii ◽  
Matthias Meurer ◽  
Chi-Ting Ho ◽  
Birgit Besenbeck ◽  
Julia Fueller ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Proteasomal Degradation ◽  
Time Range ◽  
Green Fluorescent Proteins ◽  
Red Fluorescent Proteins ◽  
Dependent Change ◽  
The Stability ◽  
Green Fluorescent

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far tFTs were constructed by combining different slower-maturing red fluorescent proteins (redFPs) with the same faster-maturing superfolder green fluorescent protein (sfGFP). Towards a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing greenFPs, while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT towards slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for design of new tFTs.

scholarly journals TnFLX: a Third-Generation mariner-Based Transposon System for Bacillus subtilis

2020 ◽  
Vol 86 (10) ◽  
Author(s):  
Felix Dempwolff ◽  
Sandra Sanchez ◽  
Daniel B. Kearns
Keyword(s):  
Antibiotic Resistance ◽  
Fluorescent Protein ◽  
Protein Translation ◽  
Delivery Vehicle ◽  
Content Type ◽  
E Coli ◽  
Vector Construction ◽  
Resistance Markers ◽  
The Stability ◽  
Green Fluorescent

ABSTRACT Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli. We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms. IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli. Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

scholarly journals The Colonization Process of Sunflower by a Green Fluorescent Protein-Tagged Isolate of Verticillium dahliae and its Seed Transmission

Plant Disease ◽  
2018 ◽  
Vol 102 (9) ◽  
pp. 1772-1778 ◽  
Author(s):  
Y. Zhang ◽  
J. Zhang ◽  
J. Gao ◽  
G. Zhang ◽  
Y. Yu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Verticillium Dahliae ◽  
Fluorescent Protein ◽  
Lateral Roots ◽  
Pollen Grains ◽  
Seed Transmission ◽  
Vascular Bundles ◽  
Long Distance ◽  
Vascular Elements ◽  
Green Fluorescent

Sunflower Verticillium wilt is a widespread and destructive disease caused by the soilborne pathogen Verticillium dahliae. To better understand the process of infection and seed transmission of the fungus, sunflower roots were inoculated with a V. dahliae strain (VdBM9-6) labeled with green fluorescent protein (GFP) and monitored microscopically. After 24 to 96 h postinoculation (hpi), conidia germinated and developed into mycelium on root hairs, elongation zones, and caps of lateral roots. Mycelium colonized vascular bundles of lateral roots and taproots at 7 days postinoculation (dpi). At 10 weeks postinoculation (wpi), the epidermal cells, cortical tissues, and vascular elements of stem, petiole, and leaf veins were colonized by mycelium. By 12 wpi, strong GFP signals were detected not only on different tissues of inflorescence but also on testa of seed and a small fraction of pollen grains. A GFP signal was not observed on cotyledon tissues in the seed. Additionally, the colonization of V. dahliae on testa was also confirmed with MNP-10 selection medium, indicating that the testa of seed is the main carrier for the long distance transmission of sunflower yellow wilt.

scholarly journals The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins

Microbiology ◽  
2006 ◽  
Vol 152 (11) ◽  
pp. 3271-3280 ◽  
Author(s):  
Jan Hänisch ◽  
Marc Wältermann ◽  
Horst Robenek ◽  
Alexander Steinbüchel
Keyword(s):  
Fusion Protein ◽  
Mycobacterium Smegmatis ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Rhodococcus Opacus ◽  
Egfp Fusion Protein ◽  
Green Fluorescent ◽  
Cell Fractions

In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli β-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia–Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1–eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1–eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1–LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of β-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.

scholarly journals Denaturation of proteins with beta-barrel topology induced by guanidine hydrochloride

2010 ◽  
Vol 24 (3-4) ◽  
pp. 367-373 ◽  
Author(s):  
Olesya V. Stepanenko ◽  
Irina M. Kuznetsova ◽  
Vladislav V. Verkhusha ◽  
Maria Staiano ◽  
Sabato D'Auria ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Binding Protein ◽  
Guanidine Hydrochloride ◽  
Odorant Binding Protein ◽  
Tryptophan Residue ◽  
Protein Classes ◽  
The Stability ◽  
Green Fluorescent ◽  
Odorant Binding

The stability of the representatives of two protein classes withβ-barrel topology: porcine odorant-binding protein (OBP) and a number of fluorescent proteins (FPs), was studied. It was shown that both of them are significantly more stable than globular α-helical andα/βproteins. At the same time the value of energy barrier between native and unfolded state for FPs exceeds that for OBP. It was found that the small guanidine hydrochloride concentrations induce local structural disturbances in proteins: changes in microenvironment of tryptophan residue in the case of odorant-binding protein and decrease in chromophore non-planarity in the case of green fluorescent protein.

scholarly journals Splitting aptamers and nucleic acid enzymes for the development of advanced biosensors

2020 ◽  
Vol 48 (7) ◽  
pp. 3400-3422 ◽  
Author(s):  
Mégane Debiais ◽  
Amandine Lelievre ◽  
Michael Smietana ◽  
Sabine Müller
Keyword(s):  
Green Fluorescent Protein ◽  
Nucleic Acid ◽  
Fluorescent Protein ◽  
Rna Aptamers ◽  
End Joining ◽  
Specific Target ◽  
Split Protein ◽  
The Subject ◽  
The Stability ◽  
Green Fluorescent

Abstract In analogy to split-protein systems, which rely on the appropriate fragmentation of protein domains, split aptamers made of two or more short nucleic acid strands have emerged as novel tools in biosensor set-ups. The concept relies on dissecting an aptamer into a series of two or more independent fragments, able to assemble in the presence of a specific target. The stability of the assembled structure can further be enhanced by functionalities that upon folding would lead to covalent end-joining of the fragments. To date, only a few aptamers have been split successfully, and application of split aptamers in biosensing approaches remains as promising as it is challenging. Further improving the stability of split aptamer target complexes and with that the sensitivity as well as efficient working modes are important tasks. Here we review functional nucleic acid assemblies that are derived from aptamers and ribozymes/DNAzymes. We focus on the thrombin, the adenosine/ATP and the cocaine split aptamers as the three most studied DNA split systems and on split DNAzyme assemblies. Furthermore, we extend the subject into split light up RNA aptamers used as mimics of the green fluorescent protein (GFP), and split ribozymes.

scholarly journals Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck

2000 ◽  
Vol 11 (5) ◽  
pp. 1585-1595 ◽  
Author(s):  
Marie-José J. E. Bijlmakers ◽  
Mark Marsh
Keyword(s):  
Tyrosine Kinases ◽  
Fluorescent Protein ◽  
Membrane Binding ◽  
Src Kinase ◽  
Direct Role ◽  
Hsp90 Activity ◽  
Src Homology ◽  
Src Homology 2 Domain ◽  
The Stability ◽  
Green Fluorescent

Tyrosine kinases of the Src family are synthesized as cytosolic proteins that subsequently translocate to membranes. Little is known of the mechanisms responsible for targeting these proteins to membranes, although a role for the cytosolic chaperone Hsp90 has been proposed. Here, we have studied the involvement of Hsp90 in the synthesis, membrane binding, and maintenance of the Src-kinase Lck. Using specific inhibitors of Hsp90, geldanamycin and radicicol, we found that functional Hsp90 is essential for the stability of newly synthesized, but not mature, Lck. Similar results were obtained for two other Src-kinases, c-Src and Lyn. In contrast, LckY505F and LckΔSH2, constitutively active Lck mutants lacking the C-terminal regulatory tyrosine or the entire Src homology 2 domain, respectively, required Hsp90 activity to stabilize the mature proteins. Lck synthesized in the absence of Hsp90 activity was degraded within 30–45 min. This unstable Lck was myristoylated normally but did not associate with membranes or CD4, interactions that normally start within minutes of the completion of Lck synthesis. A construct composed of the N-terminal unique domain of Lck fused to green fluorescent protein did not require Hsp90 activity during synthesis. In addition, this protein associated with membranes efficiently in the absence of Hsp90 activity. Together these data suggest that interaction with Hsp90 is necessary for the correct synthesis and subsequent membrane binding of Lck. However, Hsp90 does not appear to play a direct role in Lck membrane, or CD4, association.

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