title: Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker.
abstract: To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Deltaura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-5'-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Deltabop strain was constructed by transforming a Deltaura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Deltabop recombinants at a frequency of > 10-2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706, USA.
authors.forename: R F
authors.initials: RF
authors.lastname: Peck
authors.forename: S
authors.initials: S
authors.lastname: DasSarma
authors.forename: M P
authors.initials: MP
authors.lastname: Krebs
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D005819:Genetic Markers; D019617:Halobacterium salinarum / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid
chemical_list: D019843:Archaeal Proteins; D005656:Fungal Proteins; D005819:Genetic Markers; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D009964:Orotidine-5'-Phosphate Decarboxylase