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http://dx.doi.org/10.5352/JLS.2011.21.10.1370

Isolation and Characterization of Pyrimidine Auxotrophs from the Hyperthermophilic Archaeon Sulfolobus acidocaldarius DSM 639  

Choi, Kyoung-Hwa (Department of Microbiology, Pusan National University)
Cha, Jae-Ho (Department of Microbiology, Pusan National University)
Publication Information
Journal of Life Science / v.21, no.10, 2011 , pp. 1370-1376 More about this Journal
Abstract
To study the functional genomic analysis of a crenachaeon Sulfolobus acidocaldarius, we have constructed an auxotrophic mutant based on pyrEF, which encodes the pyrimidine biosynthetic enzymes orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. S. acidocaldarius was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can be selected for mutations in pyrEF genes within a pyrimidine biosynthesis cluster. Spontaneous 5-FOA-resistant mutants by ultraviolet, KH1U and KH2U, were found to contain two point mutations and a frame shift mutation in pyrE, respectively. Mutations at these sites from KH1U and KH2U decreased the activity of orotate phosphoribosyltransferase encoded by the pyrE gene and blocked the degradation of 5-FOA into toxic 5-FOMP and 5-FUMP that kill the cells. Therefore, KH1U and KH2U were uracil auxotrophs. Transformation of Sulfolobus-Escherichia coli shuttle vector pC bearing pyrEF genes from S. solfataricus P2 into S. acidocaldarius mutant KH2U restored 5-FOA sensitivity and overcame the uracil auxotrophy. This study establishes an efficient genetic strategy towards the systematic knockout of genes in S. acidocaldarius.
Keywords
Pyrimidine biosynthesis; Sulfolobus acidocaldarius; uracil auxotroph; 5-fluoroorotic acid;
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