Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.
We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 μg of vector DNA and 10(6) protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.
Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.
Steffi
S
Noack-Schönmann
Department of Biotechnology, University of Applied Sciences Jena, Carl-Zeiß-Promenade 2, Jena, 07745, Germany.
Tanja
T
Bus
Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.
Ronald
R
Banasiak
Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.
Nicole
N
Knabe
Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany.
William J
WJ
Broughton
Institute of Biology Leiden, Universiteit Leiden, Sylviusweg 72, Leiden, 2333 BE, The Netherlands.
H
H
Den Dulk-Ras
Institute of Biology Leiden, Universiteit Leiden, Sylviusweg 72, Leiden, 2333 BE, The Netherlands.
Paul Jj
PJ
Hooykaas
Department 4 (Materials & Environment), Federal Institute for Materials Research and Testing (Bundesanstalt für Material-forschung und -prüfung, BAM), Unter den Eichen 87, Berlin, 12205, Germany ; Department of Biology, Chemistry and Pharmacy & Department of Earth Sciences, Freie Universität Berlin, Malteserstrasse 74-100, Berlin, 12249, Germany.
Anna A
AA
Gorbushina
Ancestor of opportunistic pathogens & lichens; Black yeast; DNA transfer; Fungal cell-walls; Hygromycin resistance; Protoplasts; Stress-protective morphology; Sub-aerial biofilms