Genetic
transformation of the pathogenic yeast Candida parapsilosis.
Julia Zemanova (1),
Jozef Nosek (1), Lubomir Tomaska (2)
(1) Department of Biochemistry, Comenius Univ., Fac. Nat. Sci., Mlynska dolina
CH-1, Bratislava, 84215, Slovakia (Julia.Zemanova@fns.uniba.sk); (2) Department
of Genetics, Comenius Univ., Fac. Nat. Sci., Mlynska dolina B-1, 84215
Bratislava, Slovakia
The yeast Candida parapsilosis is a frequent cause of nosocomial fungemia in immunocompromised patients. Moreover, it displays several interesting features (e.g. high affinity to prosthetics, the presence of respiratory complex I in mitochondria, linear mitochondrial genome) that recently increased the interest in the study of this microorganism. To promote molecular genetic investigations of C. parapsilosis, we developed a system for genetic transformation based on the galactokinase-deficient (gal1-) mutants as recipient strains and a set of vectors containing the CpGAL1 gene coding for galactokinase as the selection marker. Moreover, we optimized protocols for transfer of plasmid DNA into C. parapsilosis cells and we compared the efficiencies of three transformation procedures, i.e. LiAc/ssDNA/PEG, electroporation and biolistic bombardment. In addition, we analyzed the nature of different vectors in transformants by Southern blot hybridization. Our results indicate that (i) circular plasmids lacking intrinsic ARS sequences are able to replicate autonomously at low copy number, but are highly unstable; (ii) the linearization of plasmid DNA prior transformation resulted in the integration of vector DNA into chromosomal GAL1 locus and increased the stability (~95%); and (iii) plasmids carrying different ARS elements isolated from C. parapsilosis genomic DNA are able to transform yeast cells with high efficiency (~103 cfu/µg) and are present in multiple copies per cell.