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.