Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.

Steffi Noack-Schönmann,Paul Jj Hooykaas,Nicole Knabe,H Den Dulk-Ras,Ronald Banasiak,William J Broughton,Anna A Gorbushina,Tanja Bus

doi:10.1186/s13568-014-0080-5

Steffi Noack-Schönmann, Paul Jj Hooykaas + Show 6 more

Open Access

https://doi.org/10.1186/s13568-014-0080-5

Copy DOI

Abstract

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.

Highlights

Melanised microcolonial fungi (MCF) are a taxonomically diverse group of Ascomycetes that colonise sub-aerial rock surfaces and other varied materials in all regions of the world, from the Antarctic to hot deserts (Staley et al 1982; Gorbushina et al 1993; Urzi et al 1995; Wollenzien et al 1995; Sterflinger and Prillinger 2001; Ruibal 2004; Selbmann et al 2005; Gorbushina 2007; Ruibal et al 2008)
A transformation system exists for N. punctiforme (e.g. Risser and Meeks, 2013) and here we report a method for stably transforming K. petricola
Different enzyme preparations containing various chitinases and β-glucanases were tested to establish an efficient protocol for the isolation of protoplasts

Summary

Introduction

Melanised microcolonial fungi (MCF) are a taxonomically diverse group of Ascomycetes that colonise sub-aerial rock surfaces and other varied materials in all regions of the world, from the Antarctic to hot deserts (Staley et al 1982; Gorbushina et al 1993; Urzi et al 1995; Wollenzien et al 1995; Sterflinger and Prillinger 2001; Ruibal 2004; Selbmann et al 2005; Gorbushina 2007; Ruibal et al 2008). SABs are principal determinants of weathering of rocks (Gorbushina and Krumbein, 2000) and other materials exposed to air (e.g. Noack-Schönmann et al 2014). SABs are important in the material sciences because they are omni-present on all exposed materials, and because they degrade the surfaces to which they adhere. The establishment of a DNA-transfer- and integration-system into K. petricola A95 will set the stage for a variety of investigations into the role of MCFs in SAB formation and biofilminduced material deterioration

Methods

Results

Conclusion