scholarly journals Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments

2010 ◽  
Vol 76 (18) ◽  
pp. 6119-6127 ◽  
Author(s):  
Putthapoom Lumjiaktase ◽  
Claudio Aguilar ◽  
Tom Battin ◽  
Kathrin Riedel ◽  
Leo Eberl
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Natural Habitat ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Broad Host Range ◽  
Construction Of Self ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria

1998 ◽  
Vol 64 (7) ◽  
pp. 2554-2559 ◽  
Author(s):  
Serina Stretton ◽  
Somkiet Techkarnjanaruk ◽  
Alan M. McLennan ◽  
Amanda E. Goodman
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Marine Bacteria ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Bacterial Species ◽  
Epifluorescence Microscopy ◽  
Broad Host Range ◽  
Green Fluorescent

ABSTRACT Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or thenpt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when thegfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed toN′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.

scholarly journals Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

2012 ◽  
Vol 140 (3) ◽  
pp. 249-266 ◽  
Author(s):  
Mehdi Najafi ◽  
Mohammad Haeri ◽  
Barry E. Knox ◽  
William E. Schiesser ◽  
Peter D. Calvert
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Diffusion ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Live Cell ◽  
Confocal Imaging ◽  
Slow Phase ◽  
Protein Diffusion ◽  
Green Fluorescent ◽  
The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

Fitness cost of the green fluorescent protein in gastrointestinal bacteria

2003 ◽  
Vol 49 (9) ◽  
pp. 531-537 ◽  
Author(s):  
Camilla Rang ◽  
James E Galen ◽  
James B Kaper ◽  
Lin Chao
Keyword(s):  
Green Fluorescent Protein ◽  
Doubling Time ◽  
Fluorescent Protein ◽  
Shigella Flexneri ◽  
Bacterial Species ◽  
Enteric Bacteria ◽  
Fitness Cost ◽  
Gfp Expression ◽  
Selection Experiments ◽  
Green Fluorescent

There are surprisingly few studies that have successfully used the green fluorescent protein (GFP) as a quantitative reporter in selection experiments screening for inducible bacterial promoters. One explanation is that GFP expression may confer a fitness cost for bacteria. To test this possibility, we monitored the doubling time in enteric bacteria expressing GFP. Four bacterial species, Escherichia coli, enterohaemorrhagic E. coli, Shigella flexneri, Salmonella typhi, and Vibrio cholerae, were examined. The level of GFP expression was varied by using a salt-inducible promoter. After accounting for the increase in doubling time resulting from elevated osmolarity, the doubling time of all bacteria was found to increase proportionally with GFP expression, and some strains were more affected than others. Cultures of the bacteria most affected by GFP exhibited a proportion of elongated cells, which suggests that GFP production could interfere with cell division in these strains. The results in this study show that GFP is costly to bacteria and suggest that overly active promoters should be difficult to obtain from a genomic promoter library. They also suggest that the chances of succeeding in using GFP as a reporter in selection experiments are increased by growing the bacteria for the fewest number of generations and by subduing the expression of GFP whenever possible, such as by using a low copy vector to clone the library.Key words: green fluorescent protein, protein cost, promoter activity.

scholarly journals Use of Green Fluorescent Protein Color Variants Expressed on Stable Broad-Host-Range Vectors to Visualize Rhizobia Interacting with Plants

2000 ◽  
Vol 13 (11) ◽  
pp. 1163-1169 ◽  
Author(s):  
Nico Stuurman ◽  
Cristina Pacios Bras ◽  
Helmi R. M. Schlaman ◽  
André H. M. Wijfjes ◽  
Guido Bloemberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Host Range ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Confocal Laser ◽  
Broad Host Range ◽  
Enhanced Yellow Fluorescent Protein ◽  
Green Fluorescent ◽  
Color Variants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

scholarly journals Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum

2002 ◽  
Vol 92 (7) ◽  
pp. 743-749 ◽  
Author(s):  
Sigal Horowitz ◽  
Stanley Freeman ◽  
Amir Sharon
Keyword(s):  
Green Fluorescent Protein ◽  
Plant Species ◽  
Fluorescent Protein ◽  
Leaf Tissue ◽  
Colletotrichum Acutatum ◽  
Signal Molecules ◽  
Primary Host ◽  
Large Numbers ◽  
Cell Layers ◽  
Green Fluorescent

Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.

Influence of Storage Temperature, Moisture Content, and Physical Impurities on the Distribution and Survival of Salmonella enterica in Poultry Fat Intended for Pet Food Use

2018 ◽  
Vol 81 (8) ◽  
pp. 1364-1372 ◽  
Author(s):  
RINARA C. KIEL ◽  
JENNIFER N. MARTIN ◽  
DALE R. WOERNER ◽  
RACHEL MURPHY ◽  
IFIGENIA GEORNARAS ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Moisture Content ◽  
Fluorescent Protein ◽  
Storage Temperature ◽  
Impurity Level ◽  
Compositional Variation ◽  
Pet Food ◽  
Poultry Fat ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Contamination of rendered products with Salmonella is a concern for the rendering industry, particularly when those products are intended for use in other foodstuffs, such as pet food. This study was conducted to understand the influence of compositional variation on the location and survivability of Salmonella in a poultry fat matrix. Specifically, this study aimed to (i) assess the influence of postinoculation time and moisture content on the distribution of Salmonella in rendered poultry fat and (ii) evaluate the impact of postinoculation time and physical parameters (i.e., impurity level and moisture content) on survival of three Salmonella strains in rendered poultry fat stored at two different temperatures. Three studies, designated as study I(a), I(b), and II, respectively, were conducted to address these objectives. In study I(a), a green fluorescent protein–expressing strain of Salmonella Typhimurium was used to map the organism within warmed (45°C) poultry fat containing various levels of moisture. In study I(b), the influence of storage temperature on the survivability of green fluorescent protein–expressing Salmonella was evaluated. In study II, the impacts of physical impurities, moisture content, and storage temperature on the survivability of three Salmonella strains (Enteritidis, Senftenberg, and Typhimurium) were assessed. The results of this study demonstrated that composition (i.e., moisture and impurity contents) influences the survivability of Salmonella in poultry fat; specifically, Salmonella is more persistent in poultry fat with a greater moisture content and water activity. Nonetheless, although composition impacts the distribution and survivability of Salmonella in poultry fat, Salmonella generally does not survive in poultry fat maintained at high temperatures (45°C and above).

scholarly journals Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds

2002 ◽  
Vol 68 (4) ◽  
pp. 1962-1971 ◽  
Author(s):  
Lawrence Stiner ◽  
Larry J. Halverson
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Broad Host Range ◽  
Bacterial Biosensor ◽  
Minimum Concentration ◽  
Fluorescence Response ◽  
Green Fluorescent ◽  
Related Compounds

ABSTRACT A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM for trichloroethylene (means ± standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di- and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

Accurate and efficient structure-based computational mutagenesis for modeling fluorescence levels of Aequorea victoria green fluorescent protein mutants

2020 ◽  
Vol 33 ◽  
Author(s):  
Majid Masso
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Classification Performance ◽  
Machine Learning Algorithms ◽  
Sequence Structure ◽  
Aequorea Victoria ◽  
Function Relationship ◽  
Green Fluorescent ◽  
The Impact ◽  
Computational Mutagenesis

Abstract A computational mutagenesis technique was used to characterize the structural effects associated with over 46 000 single and multiple amino acid variants of Aequorea victoria green fluorescent protein (GFP), whose functional effects (fluorescence levels) were recently measured by experimental researchers. For each GFP mutant, the approach generated a single score reflecting the overall change in sequence-structure compatibility relative to native GFP, as well as a vector of environmental perturbation (EP) scores characterizing the impact at all GFP residue positions. A significant GFP structure–function relationship (P < 0.0001) was elucidated by comparing the sequence-structure compatibility scores with the functional data. Next, the computed vectors for GFP mutants were used to train predictive models of fluorescence by implementing random forest (RF) classification and tree regression machine learning algorithms. Classification performance reached 0.93 for sensitivity, 0.91 for precision and 0.90 for balanced accuracy, and regression models led to Pearson’s correlation as high as r = 0.83 between experimental and predicted GFP mutant fluorescence. An RF model trained on a subset of over 1000 experimental single residue GFP mutants with measured fluorescence was used for predicting the 3300 remaining unstudied single residue mutants, with results complementing known GFP biochemical and biophysical properties. In addition, models trained on the subset of experimental GFP mutants harboring multiple residue replacements successfully predicted fluorescence of the single residue GFP mutants. The models developed for this study were accurate and efficient, and their predictions outperformed those of several related state-of-the-art methods.

Imaging of oxygen gradients in monolayer cultured cells using green fluorescent protein

2010 ◽  
Vol 299 (6) ◽  
pp. C1318-C1323 ◽  
Author(s):  
Eiji Takahashi ◽  
Michihiko Sato
Keyword(s):  
Green Fluorescent Protein ◽  
Diffusion Model ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Red Shift ◽  
Two Dimensional ◽  
Fluorescence Measurements ◽  
Hep3b Cells ◽  
Green Fluorescent ◽  
The Impact

Gradients of Po2 between capillary blood and mitochondria are the driving force for diffusional O2 delivery in tissues. Hypoxic microenvironments in tissues that result from diffusional O2 gradients are especially relevant in solid tumors because they have been related to a poor prognosis. To address the impact of tissue O2 gradients, we developed a novel technique that permits imaging of intracellular O2 levels in cultured cells at a subcellular spatial resolution. This was done, with the sensitivity to O2 ≤3%, by the O2-dependent red shift of green fluorescent protein (AcGFP1) fluorescence. Measurements were carried out in a confluent monolayer of Hep3B cells expressing AcGFP1 in the cytoplasm. To establish a two-dimensional O2 diffusion model, a thin quartz glass slip was placed onto the monolayer cells to prevent O2 diffusion from the top surface of the cell layer. The magnitude of the red shift progressively increased as the distance from the gas coverslip interface increased. It reached an anoxic level in cells located at ∼220 μm and ∼690 μm from the gas coverslip boundary at 1% and 3% gas phase O2, respectively. Thus the average O2 gradient was 0.03 mmHg/μm in the present tissue model. Abolition of mitochondrial respiration significantly dampened the gradients. Furthermore, intracellular gradients of the red shift in mitochondria-targeted AcGFP1 in single Hep3B cells suggest that the origin of tissue O2 gradients is intracellular. Findings in the present two-dimensional O2 diffusion model support the crucial role of tissue O2 diffusion in defining the O2 microenvironment in individual cells.

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