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 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 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 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 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.

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 Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a HeterologousrickAGene

2013 ◽  
Vol 80 (3) ◽  
pp. 1170-1176 ◽  
Author(s):  
Jonathan D. Oliver ◽  
Nicole Y. Burkhardt ◽  
Roderick F. Felsheim ◽  
Timothy J. Kurtti ◽  
Ulrike G. Munderloh
Keyword(s):  
Host Cell ◽  
Spotted Fever ◽  
Shuttle Vector ◽  
Negative Control ◽  
Spotted Fever Group ◽  
Content Type ◽  
Shuttle Vectors ◽  
Rickettsia Monacensis ◽  
Species Specific ◽  
Rickettsia Bellii

ABSTRACTThe rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. TherickAgene sequence and protein vary substantially betweenRickettsiaspecies, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of threeRickettsia belliivariants. These included two shuttle vector-transformedR. belliistrains and the wild-type isolate from which they were derived,R. belliiRML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one containedRickettsia monacensis-derivedrickA, and the other lacked therickAgene.Rickettsia belliitransformed to expressR. monacensis rickAhighly overexpressed this transcript in comparison to its nativerickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion ofR. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques.

Molecular biology of the pRN1 plasmid from Sulfolobus islandicus

2009 ◽  
Vol 37 (1) ◽  
pp. 42-45 ◽  
Author(s):  
Georg Lipps
Keyword(s):  
Dna Polymerase ◽  
Copy Number ◽  
Plasmid Replication ◽  
Multicopy Plasmid ◽  
Origin Of Replication ◽  
Shuttle Vectors ◽  
The Third ◽  
Double Stranded Dna ◽  
Sulfolobus Islandicus ◽  
Conserved Genes

The pRN1 plasmid is a rather small multicopy plasmid which was isolated from a Sulfolobus islandicus strain in 1993 by Wolfram Zillig and co-workers. Sequence analysis of the genome sequence suggested that three conserved genes are important for plasmid replication. These genes code for two sequence-specific DNA-binding proteins (ORF56 and ORF80) and for a large multifunctional replication protein (ORF904). The protein ORF904 has primase, DNA polymerase and helicase activity. Remarkably, the primase activity is highly sequence specific, and primers are only efficiently synthesized on templates with the motif GTG. This protein could initiate the plasmid replication by melting the double-stranded DNA at the origin of replication and by synthesizing the first primers at the replication bubble. The protein ORF56 is a repressor, and combined biochemical and genetic evidence shows that this protein is involved in regulating the copy number of the plasmid. The function of the third conserved protein, ORF80, is still mysterious. Although this protein is highly conserved, it is not essential for replication, since shuttle vectors with a deleted orf80 gene are still able to replicate in Sulfolobus. Interestingly, plasmids lacking the orf80 gene display reduced plasmid retention under non-selective conditions, raising the possibility that ORF80 is involved in plasmid partitioning or has an accessory role in plasmid replication.

scholarly journals Engineering a Hyperthermophilic Archaeon for Temperature-Dependent Product Formation

mBio ◽  
2012 ◽  
Vol 3 (2) ◽  
Author(s):  
Mirko Basen ◽  
Junsong Sun ◽  
Michael W. W. Adams
Keyword(s):  
Protein Expression ◽  
Boiling Point ◽  
Cold Shock ◽  
Pyrococcus Furiosus ◽  
Biofuel Production ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Chemical Inducers ◽  
Moderately Thermophilic ◽  
End Products

ABSTRACT Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100°C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [T opt] of 78°C) controlled by a “cold shock” promoter that is upregulated when cells are transferred from 98°C to 72°C. At 98°C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72°C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation. IMPORTANCE Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100°C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100°C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter for protein expression used in this study is activated at suboptimal growth temperatures of P. furiosus, it is a powerful genetic tool for protein expression with minimal interference of the host’s metabolism and without the need for chemical inducers.

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 Mobile Genetic Elements Related to the Diffusion of Plasmid-Mediated AmpC β-Lactamases or Carbapenemases from Enterobacteriaceae: Findings from a Multicenter Study in Spain

2015 ◽  
Vol 59 (9) ◽  
pp. 5260-5266 ◽  
Author(s):  
L. Zamorano ◽  
E. Miró ◽  
C. Juan ◽  
L. Gómez ◽  
G. Bou ◽  
...  
Keyword(s):  
Southern Hybridization ◽  
Mobile Genetic Elements ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Genetic Elements ◽  
Chromosomal Origin ◽  
Class 1 ◽  
Rapid Dissemination ◽  
Large Plasmids ◽  
Genetic Context

ABSTRACTWe examined the genetic context of 74 acquiredampCgenes and 17 carbapenemase genes from 85 of 640Enterobacteriaceaeisolates collected in 2009. Using S1 pulsed-field gel electrophoresis and Southern hybridization, 37 of 74blaAmpCgenes were located on large plasmids of different sizes belonging to six incompatibility groups. We used sequencing and PCR mapping to investigate the regions flanking the acquiredampCgenes. TheblaCMY-2-like genes were associated with ISEcp1; the surroundingblaDHAgenes were similar toKlebsiella pneumoniaeplasmid pTN60013 associated with IS26and thepspandsapoperons; and theblaACC-1genes were associated with IS26elements inserted into ISEcp1. All of the carbapenemase genes (blaVIM-1,blaIMP-22, andblaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination ofampCgenes amongEnterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons, or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons (In846 to In848) are described in this study.

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