scholarly journals The Genetic Transformation ofChlamydia pneumoniae

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Kensuke Shima ◽  
Maximilian Wanker ◽  
Rachel J. Skilton ◽  
Lesley T. Cutcliffe ◽  
Christiane Schnee ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Vector System ◽  
Species Barrier ◽  
Content Type ◽  
Green Fluorescent ◽  
Pathogenic Gene ◽  
Plasmid Backbone

ABSTRACTWe demonstrate the genetic transformation ofChlamydia pneumoniaeusing a plasmid shuttle vector system which generates stable transformants. The equineC. pneumoniaeN16 isolate harbors the 7.5-kb plasmid pCpnE1. We constructed the plasmid vector pRSGFPCAT-Cpn containing a pCpnE1 backbone, plus the red-shifted green fluorescent protein (RSGFP), as well as the chloramphenicol acetyltransferase (CAT) gene used for the selection of plasmid shuttle vector-bearingC. pneumoniaetransformants. Using the pRSGFPCAT-Cpn plasmid construct, expression of RSGFP in koala isolateC. pneumoniaeLPCoLN was demonstrated. Furthermore, we discovered that the human cardiovascular isolateC. pneumoniaeCV-6 and the human community-acquired pneumonia-associatedC. pneumoniaeIOL-207 could also be transformed with pRSGFPCAT-Cpn. In previous studies, it was shown thatChlamydiaspp. cannot be transformed when the plasmid shuttle vector is constructed from a different plasmid backbone to the homologous species. Accordingly, we confirmed that pRSGFPCAT-Cpn could not cross the species barrier in plasmid-bearing and plasmid-freeC. trachomatis,C. muridarum,C. caviae,C. pecorum, andC. abortus. However, contrary to our expectation, pRSGFPCAT-Cpn did transformC. felis. Furthermore, pRSGFPCAT-Cpn did not recombine with the wild-type plasmid ofC. felis. Taken together, we provide for the first time an easy-to-handle transformation protocol forC. pneumoniaethat results in stable transformants. In addition, the vector can cross the species barrier toC. felis, indicating the potential of horizontal pathogenic gene transfer via a plasmid.IMPORTANCEThe absence of tools for the genetic manipulation ofC. pneumoniaehas hampered research into all aspects of its biology. In this study, we established a novel reproducible method forC. pneumoniaetransformation based on a plasmid shuttle vector system. We constructed aC. pneumoniaeplasmid backbone shuttle vector, pRSGFPCAT-Cpn. The construct expresses the red-shifted green fluorescent protein (RSGFP) fused to chloramphenicol acetyltransferase inC. pneumoniae.C. pneumoniaetransformants stably retained pRSGFPCAT-Cpn and expressed RSGFP in epithelial cells, even in the absence of chloramphenicol. The successful transformation inC. pneumoniaeusing pRSGFPCAT-Cpn will advance the field of chlamydial genetics and is a promising new approach to investigate gene functions inC. pneumoniaebiology. In addition, we demonstrated that pRSGFPCAT-Cpn overcame the plasmid species barrier without the need for recombination with an endogenous plasmid, indicating the potential probability of horizontal chlamydial pathogenic gene transfer by plasmids between chlamydial species.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

scholarly journals Two-Plasmid Vector System for Independently Controlled Expression of Green and Red Fluorescent Fusion Proteins in Staphylococcus aureus

2013 ◽  
Vol 79 (9) ◽  
pp. 3133-3136 ◽  
Author(s):  
Anthony J. Brzoska ◽  
Neville Firth
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Vector System ◽  
Red Fluorescent Protein ◽  
Filament Formation ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTWe have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions inStaphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

scholarly journals A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species

2014 ◽  
Vol 81 (5) ◽  
pp. 1744-1753 ◽  
Author(s):  
Jaewook Ryu ◽  
Ukjin Lee ◽  
Jiye Park ◽  
Do-Hyun Yoo ◽  
Jung Hoon Ahn
Keyword(s):  
Abc Transporter ◽  
Recombinant Proteins ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Hydrophobic Interaction Chromatography ◽  
Target Protein ◽  
Vector System ◽  
Extracellular Medium ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTPseudomonas fluorescensis an efficient platform for recombinant protein production.P. fluorescenshas an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by insertingtliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium inP. fluorescens. In particular, AP was secreted in severalPseudomonasspecies with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such asPseudomonasspecies.

Green fluorescent protein as a reporter for gene transfer studies in the cochlea

1997 ◽  
Vol 114 (1-2) ◽  
pp. 139-147 ◽  
Author(s):  
Anil K. Lalwani ◽  
Jay J. Han ◽  
Bong J. Walsh ◽  
Sergei Zolotukhin ◽  
Nicholas Muzyczka ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

scholarly journals Gene Transfer in Rodent Nervous Tissue Following Hindlimb Intramuscular Delivery of Recombinant Adeno-Associated Virus Serotypes AAV2/6, AAV2/8, and AAV2/9

2019 ◽  
Vol 14 ◽  
pp. 117906951988902 ◽  
Author(s):  
Asad Jan ◽  
Mette Richner ◽  
Christian B Vægter ◽  
Jens R Nyengaard ◽  
Poul H Jensen
Keyword(s):  
Nervous System ◽  
Viral Load ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Adeno Associated Virus ◽  
Green Fluorescent ◽  
Virus Delivery ◽  
Intramuscular Delivery

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus–mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

scholarly journals Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard J. Smeyne ◽  
Richard A. Ashmun ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
High Titer ◽  
Hematopoietic Cells ◽  
Green Fluorescent Protein Gene ◽  
Retroviral Gene Transfer ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

scholarly journals Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus

2013 ◽  
Vol 79 (7) ◽  
pp. 2218-2224 ◽  
Author(s):  
Jeffrey L. Bose ◽  
Paul D. Fey ◽  
Kenneth W. Bayles
Keyword(s):  
Staphylococcus Aureus ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Exchange System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genetic Tools ◽  
Allelic Exchange ◽  
Green Fluorescent

ABSTRACTThebursa aurealistransposon has been used to create transposon insertion libraries ofBacillus anthracisandStaphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessentialS. aureusgenes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study ofS. aureus.

scholarly journals Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Shireen Kotay ◽  
Weidong Chai ◽  
William Guilford ◽  
Katie Barry ◽  
Amy J. Mathers
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hand Washing ◽  
Resistant Bacteria ◽  
Droplet Dispersion ◽  
Content Type ◽  
E Coli ◽  
Mode Of Transmission ◽  
Green Fluorescent

ABSTRACT There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

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