Characterization of the phosphoribosylpyrophosphate synthetase gene fromCandida albicans

Abstract

Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the transfer of phosphate from ATP to D-ribose-5-phosphate during the synthesis of purine and pyrimidine nucleotides and tryptophan and histidine biosynthesis in a variety of organisms. We cloned and sequenced the PRPP synthetase gene (PRS1) of Candida albicans because, in Saccharomyces cerevisiae, a deficiency in PRPP synthetase activity interacts with a mutation in ELM4-1 (elongated morphology) to cause constitutive pseudohyphal growth in nitrogen-rich media. In order to study the role of the C. albicans PRS1 in growth and morphogenesis, we used gene disruption to isolate PRS1 mutants; however, while heterozygous PRS1 clones were readily obtained, homozygous, null strains were not recovered indicating that PRS1 is probably essential for growth of the organism. Heterozygotes in PRS1 produced approximately 35% less PRPP synthetase (P = 0.0004) and exhibited a similar reduction in transcript levels. Confirmation of a heterozygous, single disruption in PRS1 was obtained by I-SceI digestion of chromosomal-sized DNA and Southern blot hybridizations. While no role in morphogenesis is elucidated by this work, the data strongly suggests that PRS1 is an essential gene in C. albicans and supports earlier results that indicated the presence of a single PRS gene in C. albicans.