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

Departamento de Microbiologi'a II, Facultad de Farmacia, Universidad Complutense de Madrid, Avda. Ramón y Cajal s/n, 28040 Madrid, Spain 2 Author for correspondence: J. Pla. Tel: -34 1 394 1617. Fax: +34 1 394 1745. e-mail: iesuspla@eucmax.sim.ucm.es 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 ,6 strain was obtained using the common URA3 -blaster strategy. In addition, we generated an arg5 ,6 null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans. Keywords: ARG5,6, Candida albicans , molecular biology, gene disruption, arginine