ArticleScientific reports2016
Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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9 citing papers in PubMed.
- Tn-seq of Thermus thermophilus Genome Reveals Unexpected Tolerance to Insertions in Bacterial Common Essential Genes.MicrobiologyOpen · 2026Article
- Transposon Insertion Mutagenesis in Hyperthermophilic Crenarchaeon Sulfolobus islandicus.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Global Transcriptional Programs in Archaea Share Features with the Eukaryotic Environmental Stress Response.Journal of molecular biology · 2019Review
- Random Chromosome Partitioning in the Polyploid BacteriumG3 (Bethesda, Md.) · 2019Article
- Random mutagenesis of a hyperthermophilic archaeon identified tRNA modifications associated with cellular hyperthermotolerance.Nucleic acids research · 2019Article
- Biotechnology of extremely thermophilic archaea.FEMS microbiology reviews · 2018Review
- Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.Nucleic acids research · 2018Article
- Methylation-dependent DNA discrimination in natural transformation ofProceedings of the National Academy of Sciences of the United States of America · 2017Article
- Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic CrenarchaeonArchaea (Vancouver, B.C.) · 2017Article
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7 authors.
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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.
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