A rapid seamless method for gene knockout in Pseudomonas aeruginosa

Abstract

Background: Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. METHODS: We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. RESULTS: The current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision.

Conclusion: This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods.

Keywords: Genetic knockout; Gibson assembly; Pseudomonas Aeruginosa.

Fig. 1

Construction of deletion construct via Gibson assembly. DNA fragment ends of the same color represent identical sequences, which facilitate DNA fusion. gDNA, genomic DNA

Fig. 2

a Amplification of the 500 bp upstream and downstream DNA fragments of hasS. Lane 1, 400 ng DNA ladder; lane 2, 3 μl of the 500 bp PCR product of the upstream genomic DNA sequence of hasS; lane 3, 3 μl of the 500 bp PCR product of the downstream genomic DNA sequence of hasS. b Colony PCR of fused deletion alleles. The 500 bp upstream DNA fragment and the 500 bp downstream DNA fragment were fused and cloned into the pEX18Tc vector via Gibson assembly. Lane 1, 400 ng, 100 bp DNA ladder; lane 2–6, 10 μl of PCR products of five colonies randomly selected, which all showed the 1 kbp fused deletion alleles

Fig. 3

The transformation efficiency of P. aeruginosa PAO1 with pBSP11Tc^R under various electroporation conditions. The maximum efficiency was achieved at 2.0 kV using a 1 mm gap cuvette at 4 °C or at 2.2 kV using 2 mm cuvette at 23 °C. The field strength applied at 4 °C in a 1 mm gap cuvette is 20 kV/cm. Since the membrane permeation voltage is doubled at 4 °C as compared to room temperature [17], the field strength at 23 °C in a 2 mm cuvette (11 kV/cm) is equivalent to 22 kV/cm at 4 °C. Therefore, the optimum field strength is determined to be 20–22 kV/cm (4 °C equivalent). Since the 2 mm gap cuvette is less prone to arcing, electroporation at 2.2 kV using a 2 mm gap cuvette at room temperature was used to transform the suicide pEX18Tc allelic exchange construct into P. aeruginosa PAO1. Pulse durations of 4.0 to 4.5 ms were used in most experiments

Fig. 4

Gene knockout of P. aeruginosa via homology recombination. Primer 1 and 2, universal pEX18 forward and reverse primers flanking the multiple cloning sites, respectively. Primers 3 and 4, specific to genomic DNA sequences flanking the sequences cloned into the pEX18Tc vector. Primers 1 and 2 were used to screen E. coli DH5α transformants containing the deletion allele plasmid. Primers 1 and 4 or primers 2 and 3 were used to screen merodiploids in which the deletion construct was integrated into the chromosome. Primers 3 and 4 were used to screen deletion mutants following secondary recombination

Fig. 5

Colony PCR of merodiploids. Lane 1, 400 ng 1 kB DNA ladder; lane 2, 10 μl of PCR product of a colony in which plasmid integrated via the downstream homologous sequence; lane 3, 10 μl of PCR product of a colony in which plasmid integrated via the upstream homologous sequence

Fig. 6

Screen of deletion mutants by colony PCR. Lane 1, 400 ng 1 kb DNA ladder; lane 2–15, 10 μl of PCR products of 14 colonies randomly selected from the TYS10 plate. Of the fourteen randomly selected colonies, 8 colonies were deletion mutants while six colonies reverted to wild type during the second recombination event