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Applied and Environmental Microbiology, March 2009, p. 1243-1247, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02162-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Phleomycin Increases Transformation Efficiency and Promotes Single Integrations in Schizophyllum commune{triangledown}

Arend F. van Peer, Charissa de Bekker, Arman Vinck, Han A. B. Wösten, and Luis G. Lugones*

Microbiology and Kluyver Centre for Genomics of Industrial Fermentations, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Received 18 September 2008/ Accepted 21 December 2008

Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 µg of phleomycin ml–1 after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.

* Corresponding author. Mailing address: Microbiology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. Phone: 31 30 2533016. Fax: 31 30 2513655. E-mail: l.g.lugones{at}uu.nl

{triangledown} Published ahead of print on 29 December 2008.

Applied and Environmental Microbiology, March 2009, p. 1243-1247, Vol. 75, No. 5
0099-2240/09/$08.00+0     doi:10.1128/AEM.02162-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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