title: Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica.
abstract: The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon (ΔpyrF) as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding ΔpyrF host. After positive selection of the single-crossover integration recombinants (pop-in) on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants (pop-out) with 5-fluoroorotic acid (5-FOA), the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Han
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Hua
authors.initials: H
authors.lastname: Xiang
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics*; D003470:Culture Media; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D019613:Haloarcula / Q000235:genetics*; D019623:Haloferax mediterranei / Q000235:genetics*; D008969:Molecular Sequence Data; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D014498:Uracil / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D003470:Culture Media; C407406:pyrF protein, Archaeal; D014498:Uracil; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase