Open AccessHigh frequency of double crossover recombination facilitates genome engineering in Pseudomonas aeruginosa PA14 and clone C strains
Author(s) -
Changhan Lee,
Shady Mansour Kamal,
Ute Römling
Publication year - 2019
Publication title -
microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.352
H-Index - 35
eISSN - 1465-2080
pISSN - 1350-0872
DOI - 10.1099/mic.0.000812
Subject(s) - pseudomonas aeruginosa , crossover , homologous recombination , gene , selection (genetic algorithm) , biology , genetics , clone (java method) , recombination , strain (injury) , bacteria , artificial intelligence , computer science , anatomy
Pseudomonas aeruginosa is a key opportunistic human pathogen. An established procedure to replace a target gene is two-step allelic exchange, i.e. selection of single crossover at homologous sequences and subsequent counter selection to induce double crossover for excision of the suicide vector. In this study, we found that certain strains of P. aeruginosa display a high rate of instant double crossover upon introduction of a suicide vector containing an antibiotic resistance cassette flanked by adjacent sequences for gene replacement, making the counter selection step to achieve the second crossover superfluous. Assessment of a limited panel of target genes commonly showed negligible double crossover with a frequency <20 % in the genetic reference strain PAO1, whereas a high double crossover frequency of >70 % was observed for PA14 and clone C strains. Consequently, for certain P. aeruginosa strains replacement of an ORF by a antibiotic resistance cassette can be shortened by directly selecting for double crossover recombination.