ISSN:
1365-2958
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
,
Medicine
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1046/j.1365-2958.2000.01739.x
