scholarly journals Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

2002 ◽  
Vol 68 (11) ◽  
pp. 5528-5536 ◽  
Author(s):  
Soizick Lucas ◽  
Laurent Toffin ◽  
Yvan Zivanovic ◽  
Daniel Charlier ◽  
Hélène Moussard ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Low Frequency ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Bacterial Vector ◽  
Genes Encoding ◽  
Pyrococcus Abyssi

ABSTRACT Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

scholarly journals Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

2006 ◽  
Vol 72 (1) ◽  
pp. 102-111 ◽  
Author(s):  
S.-V. Albers ◽  
M. Jonuscheit ◽  
S. Dinkelaker ◽  
T. Urich ◽  
A. Kletzin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Shuttle Vector ◽  
Sulfolobus Solfataricus ◽  
Inducible Promoter ◽  
Single Step ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Model Organisms ◽  
Hyperthermophilic Archaeon ◽  
High Level

ABSTRACT Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80°C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.

scholarly journals Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

2007 ◽  
Vol 73 (17) ◽  
pp. 5411-5420 ◽  
Author(s):  
Yu-Sin Jang ◽  
Young Ryul Jung ◽  
Sang Yup Lee ◽  
Ji Mahn Kim ◽  
Jeong Wook Lee ◽  
...  
Keyword(s):  
Succinic Acid ◽  
Shuttle Vector ◽  
Green Fluorescence Protein ◽  
Expression Vectors ◽  
Rumen Bacteria ◽  
Gene Encoding ◽  
Shuttle Vectors ◽  
Genes Encoding ◽  
Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

scholarly journals Analysis of Cytadherence-Deficient, GapA-NegativeMycoplasma gallisepticum Strain R

2000 ◽  
Vol 68 (12) ◽  
pp. 6643-6649 ◽  
Author(s):  
L. Papazisi ◽  
K. E. Troy ◽  
T. S. Gorton ◽  
X. Liao ◽  
S. J. Geary
Keyword(s):  
Shuttle Vector ◽  
Phenotypic Expression ◽  
Mycoplasma Genitalium ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Low Passage ◽  
Translational Start

ABSTRACT Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapAtranslational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapAin R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream ofgapA in the same operon and was designatedcrmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in bothMycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed thatcrmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-typegapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

scholarly journals Shuttle Vector Expression in Thermococcus kodakaraensis: Contributions of cis Elements to Protein Synthesis in a Hyperthermophilic Archaeon

2008 ◽  
Vol 74 (10) ◽  
pp. 3099-3104 ◽  
Author(s):  
Thomas J. Santangelo ◽  
L'ubomíra Čuboňová ◽  
John N. Reeve
Keyword(s):  
Translation Initiation ◽  
Affinity Purification ◽  
Shuttle Vector ◽  
Selectable Markers ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Ribosome Binding ◽  
Translation Initiation Codon ◽  
Binding Sequence

ABSTRACT Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by ΔtrpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was ∼8-fold higher than chromosome expression. An idealized ribosome binding sequence (5′-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his6) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni2+ binding and imidazole elution.

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 Shuttle Vector-Based Transformation System for Pyrococcus furiosus

2010 ◽  
Vol 76 (10) ◽  
pp. 3308-3313 ◽  
Author(s):  
Ingrid Waege ◽  
Georg Schmid ◽  
Sybille Thumann ◽  
Michael Thomm ◽  
Winfried Hausner
Keyword(s):  
Pyrococcus Furiosus ◽  
Shuttle Vector ◽  
Gel Filtration ◽  
Model Organism ◽  
Vector System ◽  
Transformation System ◽  
Reductase Gene ◽  
Genetic Transformation System ◽  
Coa Reductase ◽  
Pyrococcus Abyssi

ABSTRACT Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furio s us, but the copy number was only one to two per chromosome. This system was used for overexpression of His6-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter.

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 Defining Components of the Chromosomal Origin of Replication of the Hyperthermophilic Archaeon Pyrococcus furiosus Needed for Construction of a Stable Replicating Shuttle Vector

2011 ◽  
Vol 77 (18) ◽  
pp. 6343-6349 ◽  
Author(s):  
Joel Farkas ◽  
Daehwan Chung ◽  
Megan DeBarry ◽  
Michael W. W. Adams ◽  
Janet Westpheling
Keyword(s):  
Pyrococcus Furiosus ◽  
Shuttle Vector ◽  
Plasmid Replication ◽  
Origin Of Replication ◽  
Binding Studies ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Chromosomal Origin ◽  
Functional Components

ABSTRACTWe report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector forEscherichia coliand functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeonPyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication inP. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis andin vitrobinding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformedP. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophilePyrococcus abyssifor use inP. furiosus, plasmids based on theP. furiosuschromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.

scholarly journals Advances in Development of a Genetic System forThermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

2000 ◽  
Vol 66 (11) ◽  
pp. 4817-4821 ◽  
Author(s):  
Volker Mai ◽  
Juergen Wiegel
Keyword(s):  
Shuttle Vector ◽  
Hydrolytic Enzymes ◽  
Genetic System ◽  
Kanamycin Resistance ◽  
Shuttle Vectors ◽  
Recent Success ◽  
Kanamycin Resistance Cassette ◽  
Expression Of Genes ◽  
Expression Host ◽  
Genes Encoding

ABSTRACT Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system,Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticumJW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of theThermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.

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