Homologous Gene Knockout in the Archaeon Halobacterium Salinarum With ura3 as a Counterselectable Marker

Mol Microbiol. 2000 Feb;35(3):667-76. doi: 10.1046/j.1365-2958.2000.01739.x.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / genetics*
  • Archaeal Proteins / metabolism
  • Cloning, Molecular
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Genetic Complementation Test
  • Genetic Engineering
  • Genetic Markers
  • Halobacterium salinarum / drug effects
  • Halobacterium salinarum / genetics*
  • Halobacterium salinarum / metabolism
  • Molecular Sequence Data
  • Mutation
  • Orotic Acid / analogs & derivatives
  • Orotic Acid / pharmacology
  • Orotidine-5'-Phosphate Decarboxylase / genetics*
  • Orotidine-5'-Phosphate Decarboxylase / metabolism
  • Selection, Genetic
  • Sequence Homology, Amino Acid

Substances

  • Archaeal Proteins
  • Fungal Proteins
  • Genetic Markers
  • Orotic Acid
  • 5-fluoroorotic acid
  • Orotidine-5'-Phosphate Decarboxylase

Associated data

  • GENBANK/AF187997