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Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker
Author(s) -
Peck Ronald F.,
DasSarma Shiladitya,
Krebs Mark P.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.01739.x
Subject(s) - ura3 , biology , halobacterium salinarum , auxotrophy , plasmid , mutant , genetics , gene , locus (genetics) , microbiology and biotechnology , archaea
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 Δ ura3 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 Δ bop strain was constructed by transforming a Δ ura3 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 Δ bop 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.