Isolation and characterization of pyrimidine auxotrophs, and molecular cloning of the pyrE gene from the hyperthermophilic archaeon Pyrococcus abyssi.
Uracil auxotrophic mutants of the hyperthermophilic archaeon Pyrococcus abyssi were isolated by screening for resistance to 5-fluoro-orotic acid (5-FOA). Wild-type strains were unable to grow on medium containing 5-FOA, whereas mutants grew normally. Enzymatic assays of extracts from wild-type P. abyssi and from pyrimidine auxotrophs demonstrated that the mutants are deficient in orotate phosphoribosyltransferase (PyrE) and/or orotidine-5'-monophosphate decarboxylase (PyrF) activity. The pyrE gene of wild-type P. abyssi and one of its mutant derivatives were cloned and sequenced. This pyrE gene could serve as selectable marker for the development of gene manipulation systems in archaeal hyperthermophiles.
Watrin
L
L
Institut Universitaire Européen de la Mer, Technopôle de Brest-Iroise, Department of Marine Microbiology, Plouzané, France. watrin@univ-brest.fr
Lucas
S
S
Purcarea
C
C
Legrain
C
C
Prieur
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D019641:DNA, Archaeal; D019766:Genes, Archaeal; D008969:Molecular Sequence Data; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics*; D011743:Pyrimidines / Q000378:metabolism*; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D019641:DNA, Archaeal; D011743:Pyrimidines; D009962:Orotate Phosphoribosyltransferase