Targeted Chromosomal Knockouts in Mycoplasma pneumoniae

Targeted Chromosomal Knockouts in Mycoplasma pneumoniae ▿ † Radha Krishnakumar 1 , * , Nacyra Assad-Garcia 1 , Gwynedd A. Benders 2 , Quang Phan 1 , Michael G. Montague 1 and John I. Glass 1 1 J. Craig Venter Institute, 9704 Medical Center Dr., Rockville, Maryland 20850 2 J. Craig Venter Institute, 10355 Science Center Dr., San Diego, California 92121 ABSTRACT Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.