scholarly journals Utilization of Virus ϕCh1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii

2013 ◽  
Vol 79 (8) ◽  
pp. 2741-2748 ◽  
Author(s):  
M. Mayrhofer-Iro ◽  
A. Ladurner ◽  
C. Meissner ◽  
C. Derntl ◽  
M. Reiter ◽  
...  
Keyword(s):  
Heterologous Gene Expression ◽  
Shuttle Vector ◽  
Extreme Environments ◽  
Vector System ◽  
Structural Protein ◽  
Transformation System ◽  
Content Type ◽  
Shuttle Vectors ◽  
Modified Method ◽  
Natrialba Magadii

ABSTRACTIn the study described here, we successfully developed a transformation system for halo(alkali)philic members of theArchaea. This transformation system comprises a series ofNatrialba magadii/Escherichia colishuttle vectors based on a modified method to transform halophilic members of theArchaeaand genomic elements of theN. magadiivirus ϕCh1. The shuttle vector pRo-5, based on therepH-containing region of ϕCh1, stably replicated inE. coliandN. magadiiand in several halophilic and haloalkaliphilic members of theArchaeanot transformable so far. The ϕCh1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of ϕCh1 ORF3452, which encodes a structural protein, and the reporter genebgaHofHaloferax lucentenseinN. magadii. The new transformation/vector system will facilitate genetic studies withinN. magadiiand other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions ofN. magadiivirus ϕCh1 in their extreme environments.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Establishment of a Tractable Genetic Transformation System in Veillonella spp.

2012 ◽  
Vol 78 (9) ◽  
pp. 3488-3491 ◽  
Author(s):  
Jinman Liu ◽  
Zhoujie Xie ◽  
Justin Merritt ◽  
Fengxia Qi
Keyword(s):  
Escherichia Coli ◽  
Genetic Transformation ◽  
Shuttle Vector ◽  
Transformation System ◽  
Genetic Studies ◽  
Content Type ◽  
Genetic Transformation System

ABSTRACTWe have constructed the firstEscherichia coli-Veillonellashuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinicalVeillonellastrain. A highly transformableVeillonellastrain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for futureVeillonellagenetic studies.

A Dual-Replicon Shuttle Vector System for Heterologous Gene Expression in a Broad Range of Gram-Positive and Gram-Negative Bacteria

2018 ◽  
Vol 75 (10) ◽  
pp. 1391-1400
Author(s):  
Mingxi Hua ◽  
Jingjing Guo ◽  
Min Li ◽  
Chen Chen ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heterologous Gene Expression ◽  
Shuttle Vector ◽  
Heterologous Gene ◽  
Vector System ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Gram Negative

scholarly journals Disruption of a Type II Endonuclease (TDE0911) Enables Treponema denticola ATCC 35405 To Accept an Unmethylated Shuttle Vector

2011 ◽  
Vol 77 (13) ◽  
pp. 4573-4578 ◽  
Author(s):  
Jiang Bian ◽  
Chunhao Li
Keyword(s):  
Southern Blot ◽  
Shuttle Vector ◽  
Cell Extract ◽  
Treponema Denticola ◽  
Type Ii ◽  
Restriction Digestion ◽  
Content Type ◽  
Shuttle Vectors ◽  
Genetic Manipulations ◽  
Dna Restriction

ABSTRACTThe oral spirocheteTreponema denticolais associated with human periodontal disease.T. denticolaATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared toT. denticolaATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence ofT. denticolaATCC 35405. In this report, we hypothesize thatT. denticolaATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome ofT. denticolaATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant ofTDE0911failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulateT. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.

scholarly journals A shuttle-vector system allows heterologous gene expression in the thermophilic methanogen Methanothermobacter thermautotrophicus ΔH

2021 ◽  
Author(s):  
Christian Fink ◽  
Sebastian Beblawy ◽  
Andreas M. Enkerlin ◽  
Lucas Mühling ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Gene Expression ◽  
Positive Selection ◽  
Large Scale ◽  
Heterologous Gene Expression ◽  
Shuttle Vector ◽  
Heterologous Gene ◽  
Vector System ◽  
Physiology And Biochemistry ◽  
Methanothermobacter Thermautotrophicus ◽  
Power To Gas

AbstractThermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis), because of their short doubling times and robust growth with high growth yields. Yet, a genetic system for these model microbes was missing despite intense work for four decades. Here, we report the establishment of tools for genetic modification of M. thermautotrophicus. We developed the modular Methanothermobacter vector system, which provided shuttle-vector plasmids (pMVS) with exchangeable selectable markers and replicons for both Escherichia coli and M. thermautotrophicus. For M. thermautotrophicus, a thermostable neomycin-resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The pMVS-plasmid DNA was transferred from E. coli into M. thermautotrophicus via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assays, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus on formate as the sole growth substrate, while this was not possible for the empty vector control. These genetic tools provide the basis to investigate hypotheses from four decades of research on the physiology and biochemistry of Methanothermobacter spp. on a genetic level.Significance StatementThe world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production, but also for production of high-value platform chemicals in power-to-x processes.

scholarly journals A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH

mBio ◽  
2021 ◽  
Author(s):  
Christian Fink ◽  
Sebastian Beblawy ◽  
Andreas M. Enkerlin ◽  
Lucas Mühling ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Climate Change ◽  
Gene Expression ◽  
Carbon Emissions ◽  
Heterologous Gene Expression ◽  
Shuttle Vector ◽  
Vector System ◽  
Energy Demands ◽  
The World ◽  
Methanothermobacter Thermautotrophicus ◽  
Harmful Effects

The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change.

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 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 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 Bifidobacterium -Escherichia coli Shuttle Vector Series pKO403, with Temperature-Sensitive Replication Origin for Gene Knockout in Bifidobacterium

2022 ◽  
Author(s):  
Hend Altaib ◽  
Yuka Ozaki ◽  
Tomoya Kozakai ◽  
Kouta Sakaguchi ◽  
Izumi Nomura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cloning ◽  
Replication Origin ◽  
Gene Knockout ◽  
Shuttle Vector ◽  
Temperature Sensitive ◽  
Content Type ◽  
Shuttle Vectors ◽  
Restriction Sites

A series of Bifidobacterium - Escherichia coli shuttle vectors (pKO403- lacZ′ -Cm, pKO403- lacZ′ -Sp, pKO403- lacZ′ -p15A) were constructed based on the pKO403 backbone, which carries a temperature-sensitive replication origin. These vectors carry the lacZ′ α fragment, overhung by two facing type IIS restriction sites, for blue-white selection and seamless gene cloning.

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