Hygromycin B-resistance phenotype acquired in Paracoccidioides brasiliensis via plasmid DNA integration

Yeast cells of the human pathogenic fungus Paracoccidioides brasiliensis strain Pb01 were transformed to hygromycin B resistance using the plasmid pAN7.1. Transformation was achieved by electroporation, with intact or linearized plasmid DNA. The fungus was transformed using 200 mM manitol, 5 or 7 kV/cm field strength, 25 mu F capacitance, 400 Omega resistance, 5 mu g plasmid DNA and 10(7) yeast cells in 400 mu l, and selected in BHI medium overlaid with 30 mu g/ml hygromycin B (hygB). Mitotic stability was assessed by growing transformants on non-selective BHI medium, followed by plating on hygromycin B (30 mu g/ml). Transformants were analyzed by PCR and Southern blotting, confirming the hph gene integration into the transformants genome. A low level of stability of the integrated hph sequence in the transformant genomes was observed, probably because of the multinuclearity of P brasiliensis yeast cells.