Plasmid transformation and expression of the firefly luciferase in Microbacterium testaceum type and endophytic colonizing field strains

2008 ◽  
Vol 54 (11) ◽  
pp. 964-970 ◽  
Author(s):  
Denise K. Zinniel ◽  
Zhengyu Feng ◽  
Paul H. Blum ◽  
Raúl G. Barletta ◽  
Anne K. Vidaver
Keyword(s):  
Fluorescent Protein ◽  
Bacterial Species ◽  
Firefly Luciferase ◽  
Restriction Endonuclease Analysis ◽  
Growth Conditions ◽  
E Coli ◽  
Dna Restriction ◽  
Green Fluorescent ◽  
First Time ◽  
Endonuclease Analysis

Microbacterium testaceum is a predominant endophytic bacterial species isolated from corn and sorghum in the midwestern United States. The development of genetic transfer systems for M. testaceum may enable its use for biocontrol and other applications. The type strain (IFO 12675) and field isolates (SE017, SE034, and CE648) were grown to mid-exponential phase, concentrated (1.0 × 1011CFU·mL–1), electroporated ( Escherichia coli  – Clavibacter shuttle plasmid pDM302), and plated on TSA with 10 µg·mL–1chloramphenicol. Transformation efficiencies averaged 140 CFU·µg–1of DNA. Restriction endonuclease analysis showed that pDM302 was not altered after extraction from transformants and re-introduction into E. coli. Transformants with pDM302 were also subjected to nonselective growth conditions, with the frequency of loss after one passage being 84% for IFO 12675 and 88% for SE034. We inserted the green fluorescent protein and the firefly luciferase (FFlux) reporter genes into pDM302, confirming the expression of FFlux in IFO 12675 and SE034. The SE034 FFlux strain was recovered from inoculated corn in greenhouse studies and found to fluoresce by luminometry. These results in M. testaceum demonstrate for the first time its transformability, pDM302 replication, FFlux gene expression, and the recovery of the FFlux recombinant strain from inoculated corn.

scholarly journals A Novel Cytology-Based, Two-Hybrid Screen for Bacteria Applied to Protein-Protein Interaction Studies of a Type IV Secretion System

2002 ◽  
Vol 184 (20) ◽  
pp. 5572-5582 ◽  
Author(s):  
Zhiyong Ding ◽  
Zhenming Zhao ◽  
Simon J. Jakubowski ◽  
Atmakuri Krishnamohan ◽  
William Margolin ◽  
...  
Keyword(s):  
Cell Division ◽  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Bacterial Species ◽  
Transcription Activator ◽  
E Coli ◽  
Substrate Complex ◽  
Type Iv ◽  
Green Fluorescent

ABSTRACT DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E. coli and Agrobacterium tumefaciens. DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E. coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP. These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E. coli and A. tumefaciens. Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites. The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments. DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A. tumefaciens T-DNA transfer system, in E. coli and homotypic complexes of VirB10 in A. tumefaciens. VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives. Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.

scholarly journals P34.8 (GP37) is not essential for baculovirus replication

2001 ◽  
Vol 82 (2) ◽  
pp. 299-305 ◽  
Author(s):  
Xiao-Wen Cheng ◽  
Peter J. Krell ◽  
Basil M. Arif
Keyword(s):  
Northern Blot Analysis ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Restriction Endonuclease Analysis ◽  
Gene Encoding ◽  
Conserved Domains ◽  
Reading Frame ◽  
Viral Plaque ◽  
Green Fluorescent ◽  
Endonuclease Analysis

Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotI–XbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.

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 CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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.

scholarly journals Droplet- Rather than Aerosol-Mediated Dispersion Is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Shireen M. Kotay ◽  
Rodney M. Donlan ◽  
Christine Ganim ◽  
Katie Barry ◽  
Bryan E. Christensen ◽  
...  
Keyword(s):  
Patient Care ◽  
Sampling Methods ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Air Sampling ◽  
Multidrug Resistant ◽  
Hand Washing ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT An alarming rise in hospital outbreaks implicating hand-washing sinks has led to widespread acknowledgment that sinks are a major reservoir of antibiotic-resistant pathogens in patient care areas. An earlier study using green fluorescent protein (GFP)-expressing Escherichia coli (GFP-E. coli) as a model organism demonstrated dispersal from drain biofilms in contaminated sinks. The present study further characterizes the dispersal of microorganisms from contaminated sinks. Replicate hand-washing sinks were inoculated with GFP-E. coli, and dispersion was measured using qualitative (settle plates) and quantitative (air sampling) methods. Dispersal caused by faucet water was captured with settle plates and air sampling methods when bacteria were present on the drain. In contrast, no dispersal was captured without or in between faucet events, amending an earlier theory that bacteria aerosolize from the P-trap and disperse. Numbers of dispersed GFP-E. coli cells diminished substantially within 30 minutes after faucet usage, suggesting that the organisms were associated with larger droplet-sized particles that are not suspended in the air for long periods. IMPORTANCE Among the possible environmental reservoirs in a patient care environment, sink drains are increasingly recognized as a potential reservoir to hospitalized patients of multidrug-resistant health care-associated pathogens. With increasing antimicrobial resistance limiting therapeutic options for patients, a better understanding of how pathogens disseminate from sink drains is urgently needed. Once this knowledge gap has decreased, interventions can be engineered to decrease or eliminate transmission from hospital sink drains to patients. The current study further defines the mechanisms of transmission for bacteria that colonize sink drains.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

scholarly journals Protease-activatable biosensors of SARS-CoV-2 infection for cell-based drug, neutralisation and virological assays

2021 ◽  
Author(s):  
Pehuen Pereyra Gerber ◽  
Lidia M Duncan ◽  
Edward JD Greenwood ◽  
Sara Marelli ◽  
Adi Naamati ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Firefly Luciferase ◽  
Neutralising Antibodies ◽  
Antiviral Therapies ◽  
Viral Proteases ◽  
Green Fluorescent ◽  
High Throughput Screens ◽  
Protease Expression ◽  
In Cells

The world is in the grip of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, and there is an urgent unmet clinical need for effective antiviral therapies. Many inhibitors of viral enzymes identified in vitro have limited efficacy against viral replication in cells, but conventional plaque assays are impractical for high-throughput screens. In this study, we therefore engineer cell-based biosensors of SARS-CoV-2 infection. Our assays exploit the cleavage of specific oligopeptide linkers by SARS-CoV-2 Main or Papain-like proteases, leading to the activation of green fluorescent protein (GFP) or firefly luciferase-based reporters. First, we characterise these biosensors in cells using recombinant viral proteases. Next, we confirm their ability to detect endogenous viral protease expression during infection with wildtype SARS-CoV-2. Finally, we develop a sensitive luminescent reporter cell line, confirm that it accurately quantitates infectious SARS-CoV-2 virus, and demonstrate its utility for drug screening and titration of neutralising antibodies.

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