Targeted Chromosomal Knockouts In Mycoplasma pneumoniae
Appl Environ Microbiol. 2010 Jun 11; 76(15): 5297-9.
Most gene knockouts in mycoplasmas are achieved through labor intensive transposon mutagenesis. Here we describe a method to make targeted deletions in Mycoplasma pneumoniae using 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.