Homologous gene knockout in the archaeon Halobacterium salinarum with ura3as a counterselectable marker

To facilitate the functional genomic analysis of an archaeon, we have devel oped a homologous gene replacement strategy for Halobacterium salinarum bas ed on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-m onophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-flu oroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneo us 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) o f this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel r esults were obtained with a Delta ura3 strain constructed by gene replaceme nt and with derivatives of this strain in which ura3 replaced bop. These re sults show that H. salinarum ura3 encodes functional orotidine-5'-monophosp hate decarboxylase. To demonstrate ura3- based gene replacement, a Delta bo p strain was constructed by transforming a Delta ura3 host with a bop delet ion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 w as included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Delta bop recombinants at a frequency of >10 (-2) in both approaches. These studies establish an efficient new genetic s trategy towards the systematic knockout of genes in an archaeon.