Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions
(2014)
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 106 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.
Sub-aerial biofilms (SABs) are ubiquitous microbial communities that develop at the interface between hard surfaces and the atmosphere. Inherent SAB 'core-settlers' include phototrophic algae, cyanobacteria, heterotrophic bacteria and microcolonial fungi (MCF). SABs do not simply cover hard surfaces; they interact with them in myriads of ways and bind to the underlying substrate. Secretion of extracellular mucilage aids adhesion, while organic acids and acidic polysaccharides weather the surface. As protection against solar radiation, many members of the SAB consortia produce shielding pigments while the phototrophic inhabitants are laden with photosynthetic pigments. All absorb light of many wavelengths and in addition, the cells themselves scatter light. Both effects change the spectra of incoming radiation (including wavelengths that are converted to electricity by photovoltaic cells) and decrease its intensity. To quantify these effects on SABs as complex entities of organisms and pigments, we measured the spectral properties of model and natural biofilms transferred to glass. Here we show that SABs growing on solar panels and other substrates scatter incident radiation between 250 nm up to 1800 nm and block up to 70% of its transmission. Model biofilms have the advantage that their microbial components can be 'tuned' to resemble natural ones of different compositions thus providing a novel materials-testing tool.