Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans

Abstract

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg^-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6d strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6d null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.