Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.
Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.
Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France.|CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, 69100, Villeurbanne, France.|Univ Lyon, ENS de Lyon, CNRS UMR 5276, Lyon, France.
Natalia
N
Guschinskaya
Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France.|CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, 69100, Villeurbanne, France.
Romain
R
Brunel
Univ Lyon, ENS de Lyon, CNRS UMR 5276, Lyon, France.|Univ Lyon, INSA-Lyon, Université Claude Bernard Lyon 1, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 11 Avenuue Jean Capelle, 69621 Villeurbanne cedex, France.
Maxime
M
Tourte
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia USA.
Gina L
GL
Lipscomb
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia USA.
Michael W W
MWW
Adams
Univ Lyon, ENS de Lyon, CNRS UMR 5276, Lyon, France.|Univ Lyon, INSA-Lyon, Université Claude Bernard Lyon 1, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 11 Avenuue Jean Capelle, 69621 Villeurbanne cedex, France.
Philippe
P
Oger
Univ Lyon, Université Claude Bernard Lyon 1, INSA-Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, 10 rue Raphaël Dubois, F-69622, Villeurbanne, France.|CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ Lyon, 69100, Villeurbanne, France.
Xavier
X
Charpentier
D000818:Animals; D003470:Culture Media; D004251:DNA Transposable Elements; D015723:Gene Library; D019766:Genes, Archaeal; D020043:Genes, Essential; D023281:Genomics; D006720:Homozygote; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D010641:Phenotype; D011123:Polyploidy; D020027:Pyrococcus furiosus / Q000235:genetics*
D003470:Culture Media; D004251:DNA Transposable Elements