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 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.

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 Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression inClostridium perfringens

2010 ◽  
Vol 77 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Andrea H. Hartman ◽  
Hualan Liu ◽  
Stephen B. Melville
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Shuttle Vector ◽  
Yellow Fluorescent Protein ◽  
Inducible Promoter ◽  
Gliding Motility ◽  
Glucuronidase Activity ◽  
Atpase Gene ◽  
Regulated Gene Expression ◽  
Promoter System

ABSTRACTClostridium perfringensis a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working withC. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is anEscherichia coli-C. perfringensshuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of genebgaL(bgaR-PbgaL). To measure transcription at thebgaLpromoter in pKRAH1, theE. colireporter genegusA, encoding β-glucuronidase, was placed downstream of the PbgaLpromoter to make plasmid pAH2. When transformed into three strains ofC. perfringens, pAH2 exhibited lactose-inducible expression.C. perfringensstrain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated genebglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express theyfp-pilBgene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility inC. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in apilCmutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

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 An Lrp-Like Protein of the Hyperthermophilic Archaeon Sulfolobus solfataricus Which Binds to Its Own Promoter

1999 ◽  
Vol 181 (5) ◽  
pp. 1474-1480 ◽  
Author(s):  
Alessandra Napoli ◽  
John van der Oost ◽  
Christoph W. Sensen ◽  
Robert L. Charlebois ◽  
Mosé Rossi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sulfolobus Solfataricus ◽  
Regulation Of Gene Expression ◽  
Open Reading Frames ◽  
Tata Box ◽  
Growth Stages ◽  
Hyperthermophilic Archaeon ◽  
Gene Coding ◽  
Multiple Sequences ◽  
Transcriptional Elements

ABSTRACT Regulation of gene expression in the domain Archaea, and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea(i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea. We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus. The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria. We show that Lrs14, expressed in Escherichia coli, is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus.

A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector

2003 ◽  
Vol 48 (5) ◽  
pp. 1241-1252 ◽  
Author(s):  
Melanie Jonuscheit ◽  
Erika Martusewitsch ◽  
Kenneth M. Stedman ◽  
Christa Schleper
Keyword(s):  
Reporter Gene ◽  
Shuttle Vector ◽  
Sulfolobus Solfataricus ◽  
Hyperthermophilic Archaeon ◽  
Gene System ◽  
Reporter Gene System

scholarly journals First report of a tetracycline-inducible gene expression system for mollicutes

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Marc Breton ◽  
Evelyne Sagné ◽  
Sybille Duret ◽  
Laure Béven ◽  
Christine Citti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Inducible Promoter ◽  
High Level Expression ◽  
Spiroplasma Citri ◽  
High Level ◽  
Tetr Repressor ◽  
Promoter System ◽  
Inducible Gene

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

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