Development of an integrative transformation system for the opportunistic pathogenic yeast Candida lusitaniae using URA3 as a selection marker

Abstract

The nucleotide sequence of the URA3 gene encoding orotidine-5 -phosphate decarboxylase (OMP DCase) of the human opportunistic pathogen yeast Candida lusitaniae was determined by degenerate PCR and chromosome walking. Deduced amino acid sequence showed strong homologies (59-85% identity) with OMP DCases of different Saccharomycetales and allowed identification of the known conserved domains. Very close upstream from the URA3 gene, the 3 -end of a gene encoding a Gea2-like protein was identified. A non-revertible C. lusitaniae ura3 mutant was selected on the basis of 5-fluoroorotic acid resistance. The mutation was a single point mutation resulting in the amino acid substitution D95V in a highly conserved domain, and in a concomitant EcoRV restriction site polymorphism. The mutant strain was successfully transformed to prototrophy following electroporation with the URA3 gene cloned in an integrative vector, with frequencies of 100-200 transformants per µg of DNA. Southern blot analysis revealed that almost all transformants were derived from homologous recombination events at the resident locus. The GeneBank Accession No. for C. lusitaniae URA3 gene is AF450297.

Nonsense and Missense Mutations in FCY2 and FCY1 Genes Are Responsible for Flucytosine Resistance and Flucytosine-Fluconazole Cross-Resistance in Clinical Isolates of Candida lusitaniae

“…The pUFCY2 complementation plasmid (6.6 kb) was previously described (7). It contains the fulllength FCY2 gene (2,117 bp) subcloned into the SpeI-NsiI restriction sites of the pGEM-U plasmid containing the C. lusitaniae URA3 gene (14).…”

Differential Involvement of Histidine Kinase Receptors in Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

“…The resulting PCR amplification fragment was then successively subcloned into the SpeI-NsiI and NcoI-SacII restriction sites, located upstream and downstream, respectively, of the URA3 gene from the pGEM-U plasmid (26), to yield the plasmid pGUN, containing the REP-URA3-REP fragment (abbreviated "GUN" sequence).…”

“…The recent achievement of C. lusitaniae whole genome sequencing clearly reflects a growing interest of the scientific community in this opportunistic yeast. As a laboratory model, C. lusitaniae is an experimentally tractable haploid organism offering formal genetic tools based upon a complete sexual cycle reproducible in vitro (27,51,72) and an integrative transformation system for gene disruption using the URA3 gene as a selection marker (26,71,72).…”

Contributions of the Response Regulators Ssk1p and Skn7p in the Pseudohyphal Development, Stress Adaptation, and Drug Sensitivity of the Opportunistic Yeast Candida lusitaniae

“…Furthermore, this opportunistic yeast can efficiently switch to pseudohyphal growth (13). As a laboratory model, C. lusitaniae is an experimentally tractable haploid organism offering formal genetic tools based upon a complete sexual cycle reproducible in vitro (11,23,31) and an integrative transformation system for gene disruption (10,30,31).…”