An Improved Method for Rapid Unmarked Gene Deletion in Bacteria: Pseudomonas aeruginosa as an Example

The release and updating of whole-genome sequences of several representatives of the human opportunistic pathogen Pseudomonas aeruginosa have laid the ground for investigating the mechanisms of antibiotic resistance, biofilm formation, and virulence, while also offering opportunities for researchers to find new therapeutic targets to control this bacterium using a functional genomics approach. However, there is still a lack of detailed protocols describing gene inactivation methods in P. aeruginosa, resulting in failures and extra time spent designing in-house protocols. Here, we introduce a rapid, efficient, and unmarked deletion mutagenesis method combining overlap extension PCR, efficient conjugation, and the traditionally used sacB-based counter-selection procedure. Efficient generation of deletion mutants using this detailed protocol can be easily completed in one week using standard lab reagents. Importantly, this method may be adaptable to other bacteria where the sacB-based counter-selection system works. Key features • Using modified overlap extension PCR (MOE-PCR), the deletion construct can be generated rapidly in a single fusion PCR without intermediate gel purification. • Only one transformation step is involved in the whole protocol, thus saving time compared to marked mutagenesis involving Flp (flippase) recombinase recognition target (FRT)-mediated resolution. • Pseudomonas agar P medium (PM) and casamino acid medium (CAA) reduce reactive oxygen species (ROS)-mediated toxicity, facilitating deletion of oxidative stress-sensitive genes. • The sacB-based counter-selection works in many Gram-negative bacteria and even some Gram-positive actinobacteria, so the protocol may be adaptable to such species.