title: Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii, and its use for gene discovery.
abstract: BACKGROUND
  Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways.
  
  
  RESULTS
  Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned.
  
  
  CONCLUSIONS
  We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.
authors.affiliation: Division of Genetics and Physiology, Department of Biology, University of Turku, Turku, Finland. saija.kiljunen@utu.fi.
authors.forename: Saija
authors.initials: S
authors.lastname: Kiljunen
authors.affiliation: Division of Genetics and Physiology, Department of Biology, University of Turku, Turku, Finland. maria.pajunen@helsinki.fi.|Current address: Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland. maria.pajunen@helsinki.fi.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Pajunen
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA. kierdilks@gmail.com.
authors.forename: Kieran
authors.initials: K
authors.lastname: Dilks
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA. sstorf@yahoo.de.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Storf
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia, PA, USA. pohlschr@sas.upenn.edu.
authors.forename: Mechthild
authors.initials: M
authors.lastname: Pohlschroder
authors.affiliation: Division of Genetics and Physiology, Department of Biology, University of Turku, Turku, Finland. harri.savilahti@utu.fi.
authors.forename: Harri
authors.initials: H
authors.lastname: Savilahti
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics*; D002338:Carotenoids / Q000096:biosynthesis; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D015723:Gene Library; D018390:Gene Targeting; D056726:Genetic Association Studies; D019622:Haloferax volcanii / Q000235:genetics*; D053858:Metabolic Networks and Pathways; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D019843:Archaeal Proteins; D004251:DNA Transposable Elements; D002338:Carotenoids