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

Noack-Schönmann S, Bus T, Banasiak R, Knabe N, Broughton WJ, Den Dulk-Ras H, Hooykaas PJ, Gorbushina AA - AMB Express (2014)

Bottom Line: This mixture was equally effective on the melanin-minus mutant and the type-strain.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

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 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.

No MeSH data available.


Related in: MedlinePlus

Knufia petricolaA95 morphology. A- Being a typical MCF, K. petricola A95 retains protective pigmentation and restricted colony growth even under favourable growth conditions on a Petri dish; B - As a result of a spontaneous mutation, the A95 pink mutant (A95p) is deficient in melanin synthesis. The orange/red colour is caused by carotenoids that are normally masked by melanins. C- Micrograph of A95 cells with melanin. The ball-shaped, compact A95 cells are surrounded by a thick cell-wall and are embedded in extracellular polymeric substances that are arranged in compact clusters of cells. D- A95 protoplasts prepared from similar cells to those shown in Figure 1C. A95 protoplasts were most effectively isolated by digestion of the cell-wall using lysing enzymes from Trichoderma harzianum and prevented from bursting in a 1M KCl buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230810&req=5

Figure 1: Knufia petricolaA95 morphology. A- Being a typical MCF, K. petricola A95 retains protective pigmentation and restricted colony growth even under favourable growth conditions on a Petri dish; B - As a result of a spontaneous mutation, the A95 pink mutant (A95p) is deficient in melanin synthesis. The orange/red colour is caused by carotenoids that are normally masked by melanins. C- Micrograph of A95 cells with melanin. The ball-shaped, compact A95 cells are surrounded by a thick cell-wall and are embedded in extracellular polymeric substances that are arranged in compact clusters of cells. D- A95 protoplasts prepared from similar cells to those shown in Figure 1C. A95 protoplasts were most effectively isolated by digestion of the cell-wall using lysing enzymes from Trichoderma harzianum and prevented from bursting in a 1M KCl buffer.

Mentions: K. petricola A95 (= CBS 123872) was isolated from the surface of a marble rock near the Philopappos monument on Musaios Hill, Athens (Greece) (Gorbushina et al. [2008]). It is maintained in the culture collection of the Centraalbureau for Schimmelcultures (CBS 123872) in Utrecht and the Federal Institute for Materials Research and Testing in Berlin (A95). A spontaneous pink mutant of A95 deficient in melanin synthesis was isolated (A95p). The characteristic orange/pink colour of the mutant (Figure 1B) is caused by carotenoids (Gorbushina et al. [2008]) that are normally masked by the melanised cell-wall. The type-strain (A95) and A95p were cultivated in liquid malt-extract medium (MEB)[2% (w/v) malt extract, 0.1% (w/v) peptone, 2% (w/v) glucose] at 25°C and 100 rpm. The cultures were refreshed each week as described in Nai et al. ([2013]). A one week old culture was homogenised along with 10 steel balls (Ø 5 mm) in a ball mill (RetschTM, Retsch GmbH, 42781 Haan, Germany) for 30 s−1 and 10 min then diluted 1:100 with fresh malt extract medium.


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

Noack-Schönmann S, Bus T, Banasiak R, Knabe N, Broughton WJ, Den Dulk-Ras H, Hooykaas PJ, Gorbushina AA - AMB Express (2014)

Knufia petricolaA95 morphology. A- Being a typical MCF, K. petricola A95 retains protective pigmentation and restricted colony growth even under favourable growth conditions on a Petri dish; B - As a result of a spontaneous mutation, the A95 pink mutant (A95p) is deficient in melanin synthesis. The orange/red colour is caused by carotenoids that are normally masked by melanins. C- Micrograph of A95 cells with melanin. The ball-shaped, compact A95 cells are surrounded by a thick cell-wall and are embedded in extracellular polymeric substances that are arranged in compact clusters of cells. D- A95 protoplasts prepared from similar cells to those shown in Figure 1C. A95 protoplasts were most effectively isolated by digestion of the cell-wall using lysing enzymes from Trichoderma harzianum and prevented from bursting in a 1M KCl buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230810&req=5

Figure 1: Knufia petricolaA95 morphology. A- Being a typical MCF, K. petricola A95 retains protective pigmentation and restricted colony growth even under favourable growth conditions on a Petri dish; B - As a result of a spontaneous mutation, the A95 pink mutant (A95p) is deficient in melanin synthesis. The orange/red colour is caused by carotenoids that are normally masked by melanins. C- Micrograph of A95 cells with melanin. The ball-shaped, compact A95 cells are surrounded by a thick cell-wall and are embedded in extracellular polymeric substances that are arranged in compact clusters of cells. D- A95 protoplasts prepared from similar cells to those shown in Figure 1C. A95 protoplasts were most effectively isolated by digestion of the cell-wall using lysing enzymes from Trichoderma harzianum and prevented from bursting in a 1M KCl buffer.
Mentions: K. petricola A95 (= CBS 123872) was isolated from the surface of a marble rock near the Philopappos monument on Musaios Hill, Athens (Greece) (Gorbushina et al. [2008]). It is maintained in the culture collection of the Centraalbureau for Schimmelcultures (CBS 123872) in Utrecht and the Federal Institute for Materials Research and Testing in Berlin (A95). A spontaneous pink mutant of A95 deficient in melanin synthesis was isolated (A95p). The characteristic orange/pink colour of the mutant (Figure 1B) is caused by carotenoids (Gorbushina et al. [2008]) that are normally masked by the melanised cell-wall. The type-strain (A95) and A95p were cultivated in liquid malt-extract medium (MEB)[2% (w/v) malt extract, 0.1% (w/v) peptone, 2% (w/v) glucose] at 25°C and 100 rpm. The cultures were refreshed each week as described in Nai et al. ([2013]). A one week old culture was homogenised along with 10 steel balls (Ø 5 mm) in a ball mill (RetschTM, Retsch GmbH, 42781 Haan, Germany) for 30 s−1 and 10 min then diluted 1:100 with fresh malt extract medium.

Bottom Line: This mixture was equally effective on the melanin-minus mutant and the type-strain.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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.

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 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.

No MeSH data available.


Related in: MedlinePlus