Characterization of a double gene disruption in the LYS2 locus of the pathogenic yeast, Candida albicans.

The 4.2 kbp LYS2 gene of Candida albicans encodes the 150 kDa subunit of the 180 kDa heterodimeric enzyme alpha-aminoadipate reductase. To facilitate structural and functional studies of the LYS2 gene, in this investigation both alleles were sequentially disrupted in C. albicans CAI4. The disruptions were performed using a targeting vector that contained a 2.2 kbp portion of LYS2 from which a 600-bp fragment had been deleted and replaced with a hisG-URA3-hisG cassette. Disruption of both alleles of the LYS2 locus was confirmed by Southern hybridization with appropriate probes. To determine the physiological effect of the LYS2 disruption, cell extracts were assayed for the LYS2-encoded alpha-aminoadipate reductase activity. The parent, heterozygote and LYS2-disrupted strains exhibited significant, partial and no activity, respectively. Transformation of the LYS2-disrupted strain with the LYS2 gene reversed the mutant phenotype as shown by growth on selective media and restored alpha-aminoadipate reductase activity. These results demonstrated that the LYS2 gene of C. albicans is responsible for the synthesis of functional alpha-aminoadipate reductase.