Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker

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 Δura3 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 Δbop strain was constructed by transforming a Δura3 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 Δbop 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.