CLONING, ANALYSIS AND ONE-STEP DISRUPTION OF THE ARG5,6 GENE OF CANDIDA-ALBICANS

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in st rain EL2 (Arg(-)) using a gene library constructed in the double auton omously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homolo gy (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARC5,6 gene is r esponsible for both the acetylglutamate kinase and acetylglutamyl-phos phate reductase activities, the second and third steps of arginine bio synthesis at the mitochondria. The C. albicans ARG5,6 gene complemente d the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yea st episomal plasmid using its own regulatory signals. A set of noninte grative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 Delta strain was obtained us ing the common URA3-bIaster strategy. In addition, we generated an arg 5,6 Delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.