A pathogen such as C. albicans needs an ef®cient mechanism of iron uptake in an iron-restricted environment such as is the human body. A ferric-reductase activity regulated by iron and copper, and analogous to that in S. cerevisiae, has been described in C. albicans. We have developed an in-plate protocol for the isolation of clones that complement an aft1 mutation in S. cerevisiae that makes cells dependent on iron for growth. After transformation of S. cerevisiae aft1 with a C. albicans library, we have selected clones that grow in conditions of iron de®ciency and share an identical plasmid, pIRO1, with a 4500 bp insert containing the URA3 gene and an ORF (IRO1) responsible for the suppression of the iron dependency. IRO1 does not show homology with AFT1 or with other sequences in the databases. Northern analysis demonstrates constitutive expression of IRO1. CAI4, a C. albicans strain isolated as Dura3, also has a deletion of the 3k half of IRO1, and displays in YNB medium similar phenotypic characteristics to S. cerevisiae aft1 mutant strains. Therefore, we consider IRO1 as a gene of C. albicans involved in the utilization of iron. However, in extreme conditions of iron deprivation, CAI4 seems to activate alternative mechanisms of iron uptake that allow a better growth than the wild strain SC5314. Analysis of its predicted protein sequence is in agreement with a role of Iro1p as a transcription factor.

Aims:  This work focuses on the development of a method for the identification of pathogenic yeast. With this aim, we target the nucleotide sequence of the RPS0 gene of pathogenic yeast species with specific PCR primers. PCR analysis was performed with both the genomic DNA, whole cells of clinical isolates of Candida species and clinical samples. Methods and Results:  A single pairs of primers, deduced from the nucleotide sequence of the RPS0 gene from pathogenic yeast, were used in PCR analysis performed with both the genomic DNA and whole cells of clinical isolates of Candida species and clinical samples. The primers designed are highly specific for their respective species and produce amplicons of the expected sizes and fail to amplify any DNA fragment from the other species tested. The set of primers was tested successfully for the identification of yeast from colonies, blood cultures and clinical samples. These results indicate that genes containing intron sequences may be useful for designing species‐specific primers for the identification of fungal strains by PCR. The sensitivity of the method with genomic DNA was evaluated with decreasing DNA concentrations (200 ng to 1 pg) and different cell amounts (107–105 cells). Conclusion:  The results obtained show that the amplification of RPS0 sequences may be suitable for the identification of pathogenic and other yeast species. Significance and Impact of the Study:  Identification of Candida species using molecular approaches with high discriminatory power is important in determining adequate measures for the interruption of transmission of this yeast. The approach described in this work is based on standard technology, and it is specific, sensitive and does not involve complex and expensive equipment. Furthermore, the method developed in this work not only can be used in eight yeast species, but also provides the basis to design primers for other fungi species of clinical, industrial or environmental interest.

A pathogen such as C. albicans needs an ef®cient mechanism of iron uptake in an ironrestricted environment such as is the human body. A ferric-reductase activity regulated by iron and copper, and analogous to that in S. cerevisiae, has been described in C. albicans. We have developed an in-plate protocol for the isolation of clones that complement an aft1 mutation in S. cerevisiae that makes cells dependent on iron for growth. After transformation of S. cerevisiae aft1 with a C. albicans library, we have selected clones that grow in conditions of iron de®ciency and share an identical plasmid, pIRO1, with a 4500 bp insert containing the URA3 gene and an ORF (IRO1) responsible for the suppression of the iron dependency. IRO1 does not show homology with AFT1 or with other sequences in the databases. Northern analysis demonstrates constitutive expression of IRO1. CAI4, a C. albicans strain isolated as Dura3, also has a deletion of the 3k half of IRO1, and displays in YNB medium similar phenotypic characteristics to S. cerevisiae aft1 mutant strains. Therefore, we consider IRO1 as a gene of C. albicans involved in the utilization of iron. However, in extreme conditions of iron deprivation, CAI4 seems to activate alternative mechanisms of iron uptake that allow a better growth than the wild strain SC5314. Analysis of its predicted protein sequence is in agreement with a role of Iro1p as a transcription factor.