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Antimicrobial Agents and Chemotherapy, October 2009, p. 4429-4432, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00388-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

New Selectable Marker for Manipulating the Simple Genomes of Mycoplasma Species {triangledown}

Mikkel A. Algire, Carole Lartigue, David W. Thomas,{dagger} Nacyra Assad-Garcia, John I. Glass, and Chuck Merryman*

The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850

Received 23 March 2009/ Returned for modification 27 July 2009/ Accepted 6 August 2009

Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation—an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.

* Corresponding author. Mailing address: 9704 Medical Center Drive, Building 1, Room 322, Rockville, MD 20850. Phone: (301) 795-7281. Fax: (240) 268-4004. E-mail: cmerryma{at}jcvi.org

{triangledown} Published ahead of print on 17 August 2009.

{dagger} Present address: Biotechnology Industry Organization (BIO), 1201 Maryland Avenue SW, Washington, DC 20024.

Antimicrobial Agents and Chemotherapy, October 2009, p. 4429-4432, Vol. 53, No. 10
0066-4804/09/$08.00+0     doi:10.1128/AAC.00388-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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