Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions

Abstract

BackgroundThe lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo).ResultsHere a scarless gene modification strategy based on the Red recombination system has been developed to modify Pseudomonas genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by PBAD promoter, which was functional in P. aeruginosa as well as in other bacteria. First the target gene was substituted for the sacB-bla cassette flanked by short homology regions (50 bp), and then this marker gene cassette could be replaced by the PCR fragment flanking itself, generating target-deleted genome without any remnants and no change happened to the surrounding region. Twenty genes involved in the synthesis and regulation pathways of the phenazine derivate, pyocyanin, were modified, including one single-point mutation and deletion of two large operons. The recombination efficiencies ranged from 88% to 98%. Multiple-gene modification was also achieved, generating a triple-gene deletion strain PCA (PAO1, ΔphzHΔphzMΔphzS), which could produce another phenazine derivate, phenazine-1-carboxylic acid (PCA), efficiently and exclusively.ConclusionsThis lambda Red-based technique can be used to generate scarless and sequential gene modification mutants of P. aeruginosa efficiently, using one-step PCR product flanked by short homology regions. Single-point mutation, scarless deletion of genes can be achieved easily in less than three days. This method may give a new way to construct genetically modified P. aeruginosa strains more efficiently and advance the regulatory network study of this organism.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…Unfortunately, the collection of genetic tools available in Pseudomonads is limited in scope and, when used in conjunction, is subject to bottlenecks that hinder the generation of a biotechnological chassis (Martínez‐García and de Lorenzo, 2011; Martínez‐García et al ., 2011, 2014). Efforts at recombineering in Pseudomonas species have been attempted with limited results (Liang and Liu, 2010; Swingle et al ., 2010). One study has isolated a ssDNA annealing recombinase (termed Ssr) demonstrating β‐like activity in P. putida (Aparicio et al ., 2016).…”

Site-Directed Mutagenesis Using Oligonucleotide-Based Recombineering

“…The length of the homology arms required for efficient recombination depends on the organism. For example, 36-50 bp (base pairs) were shown to work for E. coli or Salmonella enterica (Datsenko et al, 2000, Hansen-Wester et al, 2002 and 50-100 bp for Pseudomonas aeruginosa (Liang et al, 2010). Much longer homology arms were required for efficient Red recombination in Y. pseudotuberculosis and Y. pestis (~500 bp, Derbise et al, 2003, Sun et al, 2008 and Vibrio cholerae (100-1000 bp, Yamamoto et al, 2009).…”

“…λ Red recombination has been successfully used for convenient generation of gene deletions in E. coli (Datsenko et al, 2000), Salmonella enterica (Hansen-Wester et al, 2002), Pseudomonas aeruginosa (Liang et al, 2010, Quénée et al, 2005, Streptomyces coelicolor (Gust et al, 2004), Shigella spp. (Ranallo et al, 2006), Yersinia pseudotuberculosis (Derbise et al, 2003) and Y. pestis (Sun et al, 2008).…”

“…In these organisms, the λ Red system was used to recombine the targeting constructs with homology extensions of 50-100 bp (Liang et al, 2010) or ~500 bp (Sun et al, 2008) flanking the cassettes.…”

“…Linear targeting constructs harboring sacB combined with a kanamycin resistance gene (neo, Zhang et al, 1998), an ampicillin resistance gene (bla, Liang et al, 2010) or a chloramphenicol resistance gene (cat, Sun et al, 2008) were used for the first recombination rounds. An interesting marker combining the ability for positive selection and counterselection in one fusion gene is sacB-neo.…”