title: New selectable marker for manipulating the simple genomes of Mycoplasma species.
abstract: 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.
authors.affiliation: The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 20850, USA.
authors.forename: Mikkel A
authors.initials: MA
authors.lastname: Algire
authors.forename: Carole
authors.initials: C
authors.lastname: Lartigue
authors.forename: David W
authors.initials: DW
authors.lastname: Thomas
authors.forename: Nacyra
authors.initials: N
authors.lastname: Assad-Garcia
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.forename: Chuck
authors.initials: C
authors.lastname: Merryman
mesh_terms: D000123:Acetyltransferases / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D016680:Genome, Bacterial / Q000235:genetics*; D008957:Models, Genetic; D009174:Mycoplasma / Q000187:drug effects / Q000235:genetics*; D011691:Puromycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D011691:Puromycin; D000123:Acetyltransferases; C045336:puromycin N-acetyltransferase
references.citation: Plasmid. 1994 May;31(3):308-11
references.pmid: 8058824
references.citation: Microbiology (Reading). 2006 Feb;152(Pt 2):519-527
references.pmid: 16436439
references.citation: Nucleic Acids Res. 1981 Aug 11;9(15):3641-6
references.pmid: 6169010
references.citation: Plasmid. 2002 Sep;48(2):149-59
references.pmid: 12383732
references.citation: Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):425-30
references.pmid: 16407165
references.citation: Science. 2008 Feb 29;319(5867):1215-20
references.pmid: 18218864
references.citation: Int J Syst Evol Microbiol. 2009 Jun;59(Pt 6):1353-8
references.pmid: 19502315
references.citation: Proc Natl Acad Sci U S A. 1990 Aug;87(15):5589-93
references.pmid: 2198567
references.citation: J Bacteriol. 1985 Dec;164(3):1094-9
references.pmid: 2999069
references.citation: Plasmid. 1991 Sep;26(2):108-15
references.pmid: 1661012
references.citation: Nat Methods. 2007 Mar;4(3):251-6
references.pmid: 17293868
references.citation: Science. 1977 Mar 4;195(4281):892-4
references.pmid: 841314
references.citation: Science. 1999 Dec 10;286(5447):2165-9
references.pmid: 10591650
references.citation: Infect Immun. 2000 Dec;68(12):6643-9
references.pmid: 11083776
references.citation: FEMS Immunol Med Microbiol. 1996 Oct;15(4):199-211
references.pmid: 8908481
references.citation: Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15440-5
references.pmid: 14657399
references.citation: Cell. 2004 May 28;117(5):589-99
references.pmid: 15163407
references.citation: Mol Microbiol. 2003 Nov;50(4):1199-213
references.pmid: 14622409
references.citation: Front Biosci. 2007 Jan 01;12:2020-8
references.pmid: 17127440
references.citation: Science. 2007 Aug 3;317(5838):632-8
references.pmid: 17600181
references.citation: Plasmid. 1993 Sep;30(2):170-5
references.pmid: 8234492
references.citation: Plasmid. 2002 Mar;47(2):129-37
references.pmid: 11982334
references.citation: Nucleic Acids Res. 2003 Nov 15;31(22):6610-8
references.pmid: 14602921
references.citation: J Bacteriol. 1994 Jul;176(14):4459-62
references.pmid: 8021232