1. Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation
- Author
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Gina L. Lipscomb, Xavier Charpentier, Maxime Tourte, Natalia Guschinskaya, Philippe Oger, Michael W. W. Adams, Romain Brunel, Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Transfert de gène horizontal chez les bactéries pathogènes – Horizontal gene transfer in bacterial pathogens, Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Géologie de Lyon - Terre, Planètes, Environnement [Lyon] (LGL-TPE), Université de Lyon-Université de Lyon-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), University of Georgia [USA], Cheval, Christelle, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École normale supérieure - Lyon (ENS Lyon), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Géologie de Lyon - Terre, Planètes, Environnement (LGL-TPE), and Université de Lyon-Université de Lyon-Institut national des sciences de l'Univers (INSU - CNRS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
0301 basic medicine ,Transposable element ,Insecta ,[SDV.IMM] Life Sciences [q-bio]/Immunology ,030106 microbiology ,Mutagenesis (molecular biology technique) ,Biology ,Article ,Genes, Archaeal ,Transposition (music) ,Polyploidy ,03 medical and health sciences ,Essential ,Insertional ,Animals ,Genomic library ,Gene Library ,Genetics ,[SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Multidisciplinary ,Genes, Essential ,Homozygote ,Genomics ,Hydrogen-Ion Concentration ,biology.organism_classification ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Thermococcales ,Culture Media ,Pyrococcus furiosus ,genomic DNA ,Mutagenesis, Insertional ,030104 developmental biology ,Phenotype ,Genes ,Archaeal ,Mutagenesis ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,DNA Transposable Elements ,[SDV.IMM]Life Sciences [q-bio]/Immunology ,Transposon mutagenesis ,[SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology - Abstract
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.
- Published
- 2016
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