scholarly journals Novel Cassette-Based Shuttle Vector System for Gram-Positive Bacteria

2004 ◽  
Vol 70 (10) ◽  
pp. 6076-6085 ◽  
Author(s):  
Emmanuelle Charpentier ◽  
Ana I. Anton ◽  
Peter Barry ◽  
Berenice Alfonso ◽  
Yuan Fang ◽  
...  
Keyword(s):  
Copy Number ◽  
Modular Design ◽  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Gene Dosage ◽  
Shuttle Vector ◽  
Vector System ◽  
Shuttle Vectors ◽  
Site Specific Integration ◽  
Green Fluorescent

ABSTRACT Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P cad -cadC and constitutive P blaZ promoters were designed and analyzed in transcriptional fusions to the staphylococcal β-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

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 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.

scholarly journals New rfp- and pES213-Derived Tools for Analyzing Symbiotic Vibrio fischeri Reveal Patterns of Infection and lux Expression In Situ

2006 ◽  
Vol 72 (1) ◽  
pp. 802-810 ◽  
Author(s):  
Anne K. Dunn ◽  
Deborah S. Millikan ◽  
Dawn M. Adin ◽  
Jeffrey L. Bose ◽  
Eric V. Stabb
Keyword(s):  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Euprymna Scolopes ◽  
Shuttle Vectors ◽  
Visual Markers ◽  
Experimental Approaches ◽  
Green Fluorescent ◽  
Fluorescent Tag ◽  
Derivatives Of

ABSTRACT Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by ∼99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.

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 Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, andcfp Marker Plasmids

2000 ◽  
Vol 66 (12) ◽  
pp. 5426-5436 ◽  
Author(s):  
William G. Miller ◽  
Anne H. Bates ◽  
Sharon T. Horn ◽  
Maria T. Brandl ◽  
Marian R. Wachtel ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Promoter Sequence ◽  
Broth Culture ◽  
Single Strain ◽  
Shuttle Vectors ◽  
Chicken Skin ◽  
Green Fluorescent ◽  
Fluorescent Protein Reporter

ABSTRACT We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), orcfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacterpromoter sequence (Pc). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the Pc fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the Pc fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

scholarly journals Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity

2003 ◽  
Vol 69 (7) ◽  
pp. 3932-3937 ◽  
Author(s):  
Spencer V. Nyholm ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Signal Molecules ◽  
Natural Seawater ◽  
Gram Negative Bacteria ◽  
Light Organ ◽  
Bacterial Cells ◽  
Euprymna Scolopes ◽  
Gram Negative ◽  
Green Fluorescent

ABSTRACT Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 103 to 106 V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 106 nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.

scholarly journals Modification of Tobacco rattle virus RNA1 to Serve as a VIGS Vector Reveals That the 29K Movement Protein Is an RNA Silencing Suppressor of the Virus

2013 ◽  
Vol 26 (5) ◽  
pp. 503-514 ◽  
Author(s):  
Xianbao Deng ◽  
Jani Kelloniemi ◽  
Tuuli Haikonen ◽  
Anssi L. Vuorinen ◽  
Paula Elomaa ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Movement Protein ◽  
Tobacco Rattle Virus ◽  
Heterologous Gene ◽  
Vector System ◽  
Virus Induced Gene Silencing ◽  
Silencing Suppression ◽  
Green Fluorescent ◽  
In Cis ◽  
Pds Gene

Tobacco rattle virus (TRV) has a bipartite, positive-sense single-stranded RNA genome and is widely used for virus-induced gene silencing (VIGS) in plants. RNA1 of TRV that lacks the gene for the cysteine-rich 16K silencing-suppression protein infects plants systemically in the absence of RNA2. Here, we attempted to engineer RNA1 for use as a VIGS vector by inserting heterologous gene fragments to replace 16K. The RNA1 vector systemically silenced the phytoene desaturase (PDS) gene, although less efficiently than when the original VIGS vector system was used, which consists of wild-type RNA1 and engineered RNA2 carrying the heterologous gene. Infectious RNA1 mutants with a dysfunctional 16K suppressed silencing and enhanced transgene expression in green fluorescent protein-transgenic Nicotiana benthamiana following inoculation by agroinfiltration, unlike mutants that also lacked 29K, a movement protein (MP) gene. The 30K MP gene of Tobacco mosaic virus complemented in cis the movement defect but not the silencing suppression functions of TRV 29K. Silencing suppression by 29K occurred in the context of RNA1 replication but not in an agroinfiltration assay which tested 29K alone for suppression of sense-mediated silencing. Both 29K and 16K were needed to avoid necrotic symptoms in RNA1-infected N. benthamiana. The results shed new light on virulence factors of TRV.

scholarly journals Shuttle Vector System forMethanococcus maripaludiswith Improved Transformation Efficiency

2011 ◽  
Vol 77 (7) ◽  
pp. 2549-2551 ◽  
Author(s):  
Alison D. Walters ◽  
Sarah E. Smith ◽  
James P. J. Chong
Keyword(s):  
Transformation Efficiency ◽  
Shuttle Vector ◽  
Fold Increase ◽  
Open Reading Frame ◽  
Vector System ◽  
Methanococcus Maripaludis ◽  
Reading Frame ◽  
Shuttle Vectors

ABSTRACTWe have identified an open reading frame and DNA element that are sufficient to maintain shuttle vectors inMethanococcus maripaludis. Strain S0001, containing ORF1 from pURB500 integrated into theM. maripaludisgenome, supports a significantly smaller shuttle vector, pAW42, and a 7,000-fold increase in transformation efficiency for pURB500-based vectors.

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.

Fluorescent Antenna Proteins from the Bioluminescent Bacteria

1998 ◽  
Vol 4 (S2) ◽  
pp. 1002-1003
Author(s):  
John Lee
Keyword(s):  
Green Fluorescent Protein ◽  
Emission Spectrum ◽  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Photobacterium Phosphoreum ◽  
Bacterial Luciferase ◽  
Bioluminescent Bacteria ◽  
Photobacterium Leiognathi ◽  
Antenna Proteins ◽  
Green Fluorescent

The emission spectrum from bioluminescent bacteria has been observed to depend on the type of bacteria. Photobacterium phosphoreum species usually show bioluminescence maxima around 472 nm and Photobacterium leiognathi species to slightly longer wavelength. A certain strain (Yl) of Vibrio fischeri, has a yellow bioluminescence with maximum at 542 nm. These differences have been explained as due to the bioluminescence originating from the fluorescence transition of an “antenna” protein, participating in the bioluminescence reaction along with the enzyme bacterial luciferase. The bioluminescence from a number of coelenterates involves a similar participation of an antenna protein, the famous “Green-Fluorescent Protein” being the origin of the bioluminescence emission from these organisms.

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