An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

Abstract

Background: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.

Conclusion: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Figure 1

Map of the Gateway-compatible pEX18ApGW. This vector was derived by cloning a Gateway conversion fragment (grey) into the multiple cloning site of pEX18Ap (black). Only selected restriction enzyme cleavage sites are shown. Abbreviations: attR1 and attR2, bacteriophage λ recombination sites; bla, β-lactamase-encoding gene; cat, chloramphenicol acetyl transferase-encoding gene; ccdB, gene encoding gyrase-modifying enzyme (CcdB poisons host DNA gyrase by forming a covalent complex with the DNA gyrase A subunit and thus serves as a counter-selectable marker in gyrA⁺ cloning hosts [25]); ori, ColE1-derived replication origin; oriT, origin of conjugal transfer; sacB, Bacillus subtilis levansucrase-encoding gene. The sequence of this plasmid was deposited in GenBank and assigned accession number AY928469.

Figure 2

Schematic illustration of mutant fragment generation by overlap extension PCR. During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW-attB1 and GW-attB2, resulting in a recombination-proficient mutant PCR fragment.

Figure 3

PCR amplification of the Gm FRT cassette, PA1520 gene fragments and the overlap extension product. A. First round PCR fragments. The left panel illustrates amplification of the 1,053-bp Gm^r fragment from pPS856 which contains 24 bp (right) and 25 bp (left) overlaps with the PA1520' and 'PA1520 fragments (blue boxes). The right panel illustrates amplification of the PA1520' and 'PA1520 fragments. The 5' fragment contains 388 bp of chromosomal DNA, 25 bp overlapping the left side of the Gm^r fragment and 16 bp overlapping the GW-attB1 primer. Similarly, the 3' fragment contains 236 bp of chromosomal DNA, 24 bp overlapping the right side of the Gm^r fragment and 16 bp overlapping the GW-attB2 primer. The sequences of the gene-specific and common primers are listed in Table 1. B. Second round PCR. The purified fragments shown in panel A were used in the second round PCR illustrated in Fig. 2 to derive the indicated attB1-PA1520'-FRT-Gm-FRT-'PA1520-attB2 fragment. The entire 50 μl second round PCR reaction was subjected to agarose gel electrophoresis. The desired DNA fragment constituting the major product marked by the arrow was excised from the gel, purified and then used for the BP clonase reaction. Lanes labeled M in both panels contained Hi-Lo molecular size markers from Minnesota Molecular (Minneapolis, MN).

Figure 4

Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome. The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221-Gene::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW-Gene::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB-mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.

Figure 5

PCR analysis of marked and unmarked P. aeruginosa PA1520 deletion strains. Colony PCR was performed on either wild-type PAO1 and its PA1520 mutant derivatives, either containing a marked (PA1520::FRT-Gm-FRT) or unmarked (PA1520::FRT) PA1520 deletion. The sizes of the expected PCR fragments are indicated. Note that the short deletion removes 41 bp of the PA1520 coding sequence, corresponding to codons 131 to 145, but replaces these sequences with a 85 bp FRT scar. Lane M contained Hi-Lo molecular size markers of the indicated sizes from Minnesota Molecular.

Figure 6

Effect of deletions of P. aeruginosa GntR homologs on fabA expression. The indicated genes were deleted in strain PAO1 containing a chromosomally integrated fabA'-lacZ fusion (labelled PAO1). The control was PAO1 with a fabAΔ30'-lacZ fusion; this strain harbours the same fabA'-lacZ fusion but contains a deletion of the putative 30 nucleotide activator-binding site in the fabA promoter region. Strains were grown to mid-log phase in LB medium containing 0.05% Brij 58 +/- 0.05% oleic acid and β-galactosidase expression was monitored in triplicate samples. The dotted line marks expression levels observed in the putative activator binding-site mutant and similar levels would be expected in an activator mutant.