Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa
© Lesic and Rahme; licensee BioMed Central Ltd. 2008
Received: 28 September 2007
Accepted: 04 February 2008
Published: 04 February 2008
The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment that was obtained with a 1- or 3-step PCR protocol and that carries an antibiotic cassette flanked by a region homologous to the target locus into a strain that expresses the lambda Red recombination system.
This system has been modified for use in Pseudomonas aeruginosa. Chromosomal DNA deletions of single genes were generated using 3-step PCR products containing flanking regions 400–600 nucleotides (nt) in length that are homologous to the target sequence. A 1-step PCR product with a homologous extension flanking region of only 100 nt was in some cases sufficient to obtain the desired mutant. We further showed that the P. aeruginosa strain PA14 non-redundant transposon library can be used in conjunction with the lambda Red technique to rapidly generate large chromosomal deletions or transfer mutated genes into various PA14 isogenic mutants to create multi-locus knockout mutants.
The lambda Red-based technique can be used efficiently to generate mutants in P. aeruginosa. The main advantage of this procedure is its rapidity as mutants can be easily obtained in less than a week if the 3-step PCR procedure is used, or in less than three days if the mutation needs to be transferred from one strain to another.
The availability of an increasing number of sequenced genomes has generated the need for the development of efficient methods that will enable functional analysis of newly identified genes. The construction of knockout mutants by gene replacement has traditionally been a time consuming process because it requires several subcloning steps. A more time efficient mutagenesis method that does not require cloning was developed recently and has been used in various bacteria and fungi [1–3]. The methodology was first described in E. coli  and Aspergillus nidulans ; and subsequently applied successfully to Yersinia [4, 5], Salmonella [6, 7], Shigella  and Serratia .
The procedure involves the deletion of chromosomal genes via homologous recombination between the chromosomal region of interest and a polymerase chain reaction (PCR)-product that contains an antibiotic cassette flanked by a region of homology with the target DNA. In E. coli, a linear DNA is obtained in a 1-step PCR using primers that contain a region that is homologous with a 36-nucleotide (nt) region of the target gene. An efficient recombination between the PCR product and the chromosome is achieved by induction of the lambda phage Red operon. The Red operon encodes the nuclease inhibitor Redγ(gam) and the site specific recombinases Redα(exo) and Redβ(bet), which mediate homologous recombination . Although molecular biology techniques used in E. coli are often applicable to other bacteria, adaptations are frequently required. For example in Yersinia pseudotuberculosis, 55-nt homology extensions were generally not sufficient to allow recombination, while Y. pseudotuberculosis PCR products with extensions of approximately 500-nt, generated using a 3-step PCR procedure, could trigger reproducibly gene disruption .
Here we describe an adaptation of the lambda Red-based methodology for use with P. aeruginosa, in which chromosomal single gene deletions were generated using a 3-step PCR product containing 600- to 400-nt flanking regions that are homologous to the target sequence. We further examined the feasibility of using 1-step PCR product with only 100-nt homology extension for obtaining mutants. Finally we tested the ability of this method to delete large chromosomal regions.
Gene disruption using a 3-step PCR product
Gene disruption using a 1-step PCR product
The mutagenesis process could be simplified by using a PCR fragment with a shorter region of homology to the target gene. This would enable the product to be obtained in a 1-step PCR using long primers that are homologous to the kanamycin resistance cassette at their 3' ends and homologous to the target gene at their 5' ends. Thus in order to reduce the length of homologous extension necessary to select for P. aeruginosa recombinants, the following four sets of primers were designed to amplify the pqsC::kan locus: F-pqsC600/R-pqsC600, F-pqsC300/R-pqsC300, F-pqsC150/R-pqsC150 and F-pqsC100/R-pqsC100 (Additional file 2). The PCR products generated contained the antibiotic cassette flanked with 600, 300, 150 or 100-nt homologous regions to the pqsC-surrounding region; when 8 μg of each product was electroporated, 35, 50, 60 and 40 KmR colonies were obtained, respectively, and out of those 30, 46, 50 and 34 positive recombinants were identified based on their inability to produce pyocyanin, which corresponds to a 83–92% positive rate. Five recombinants from each transformation event were analyzed by PCR and the expected products were obtained systematically using primers Fupout-pqsC/Rinkan and Fupout-pqsC/Rdownout-pqsC (Additional file 2). These results show that in the case of pqsC mutagenesis the number of positive recombinants was not dependent on the length of the homologous region used and that a 100-nt homologous sequence was sufficient to allow recombination.
To further test the feasibility of 1-step PCR for this application, we designed and purchased primers (Sigma Aldrich) that contain at their 5' extremity 100-nt homology to the flanking regions of kynBU ( F-kynBU100-kan/R-kynBU100-kan) or lasR (F-lasR100-kan/R-lasR100-kan) and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette (Additional file 2). The kanamycin resistance cassette was amplified using pUC4K as the template and a mixture of two sets of primers: F100-kan/R100-kan and Fup/Rdown (Fig 1B). The resultant PCR product was used for electroporation. ΔkynBU recombinants, but not ΔlasR mutants, were obtained using 8 μg of PCR product and confirmed by PCR as described above. Our inability to obtain the ΔlasR mutant with this protocol indicates that the 1-step PCR method cannot be considered highly reliable and that 100 nt is likely close to the minimal length of homology needed.
Transfer of deletion to other mutant backgrounds
The lambda Red methodology was combined with 1-step PCR to transfer a knockout mutation from the original strain in which it was generated to other strains. We amplified the lasR::kan locus using the DNA of the previously obtained mutant as the template and the primer pair F-lasR/R-lasR. The amplification generated a PCR product containing 490-nt and 445-nt upstream and downstream flanking target regions. Eight-microgram aliquots of PCR product were electroporated into three PA14 isogenic mutants (ΔmvfR, ΔpqsE and ΔpqsA), and all three strains successfully acquired the mutated locus, which was demonstrated by DNA amplification using primer pairs Fupout-lasR/Rinkan and Fupout-lasR/Rdownout-lasR.
A comprehensive non-redundant PA14 transposition mutant library was recently constructed by random insertion of the MAR2xT7 transposon which confers gentamycin-resistance . We tested whether the lambda Red methodology would allow the transfer of loci where the MAR2xT7 transposon has been randomly inserted. The gentamycin cassette that had been inserted into pqsC was amplified using primers F-pqsC600/R-pqsC600 to generate a product with 379-nt upstream and 471-nt downstream pqsC-surrounding regions. Similar to the deletion experiments above, 2, 4 or 8 μg samples of DNA were electroporated into PA14/pUCP18-RedS and yielded 14, 14 and 50 GmR recombinants, respectively. Five recombinants were tested for the inability to produce pyocyanin in cultures and the presence of the MAR2xT7 transposon was checked by PCR amplification using primers F-pqsC600/R-pqsC600, respectively (data not shown). These results demonstrate that the lambda Red technique can be used in conjunction with a mutant library to achieve rapid transfer of mutated genes into various PA14 isogenic mutants and thereby enable the creation of multi-locus knockout mutants.
Deletion of a large DNA region using the lambda Red system
Lambda Red-based methodology has the potential of enabling large regions of chromosomal DNA, including large gene clusters and operons and pathogenicity islands, to be deleted. We examined whether the lambda Red system could be also used to delete the entire P. aeruginosa HSI-II locus , an approximately 24 kb genomic region encoding a putative type VI secretion system located between PA14_43110 and PA14_42880 genes. The PCR procedure followed to obtain the PCR product necessary to delete the HSI-II locus is presented in Fig 1C. First, genomic DNA of mutants of the PA14 non-redundant transposon library: PA14_43100::MAR2xT7 and PA14_42880::MAR2xT7 were used as template to generate the PCR fragment containing the gentamycin cassette flanked by the HSI-II locus-borders. Primers Fup-PA14_43100/Rup-MAR2xT7 were designed to amplify MAR2xT7 and the 890 nt upstream sequence when inserted into PA14_43100; while Fdown-MAR2xT7/Rdown-PA14_42880 were used to amplify MAR2xT7 and the 955 nt downstream sequence when inserted into PA14_42880 (Additional file 2). In a second PCR-step, the resulting upstream and downstream fragments were mixed with the primers Fup-PA14_43100 and Rdown-PA14_42880 and a PCR product containing MAR2xT7 flanked by the borders of the HSI-II locus obtained. PCR product aliquots of 2, 4 or 8 μg were electroporated into PA14/pUCP18-RedS, and 256, 280 and 300 recombinants were selected on gentamycin, respectively. All mutants tested were producing pyocyanin (Fig 2). DNA amplification using primers Fup-PA14_43100/Rup-MAR2xT7 and Fup-PA14_43100/Rdown-PA14_42880 confirmed deletion of the target region in 5 independent colonies; Fig 4B shows the PCR products obtained for one representative recombinant. While no DNA amplification with the primers Fup-PA14_43100/Rdown-PA14_42880 flanking the HSI-II locus was obtained when wild type genomic DNA was used as a template, a fragment of 2844 bp was amplified when mutant genomic DNA was used (Fig 4B).
The principal advantage of lambda Red-based mutagenesis is its rapidity. The technique can be performed in less than a week if the 3-step PCR procedure is used, and in less than three days if just the transfer of an existing mutated locus is needed. The protocol has been also further simplified by the use of Choi et al's method for preparing electrocompetent cells .
Our results demonstrated that 100-nt homology between the PCR product and the target gene is sufficient in some cases to produce recombination. Nevertheless to maximize the chances of obtaining mutants, the 3-step PCR technique should be used to generate a product with 600- to 400-nt homology extension to the target gene. Also, note that fragments of the same length may have different efficiency of recombination rates due to differing sequence contexts.
The technique has yet never failed; however further experiments should be conducted to additionally test the ability of this methodology to transfer genes marked with an antibiotic cassette from PA14 to various non-isogenic P. aeruginosa strains.
To simplify the process, transposon mutants in the PA14 transposon mutant library can serve as templates for amplification of genes of interest already interrupted by an inserted MAR2xT7 transposon carrying the gentamycin cassette. We routinely use this method to produce multi-locus knockout mutants. Moreover, PA14 transposon mutant library can also serve for the rapid deletion of large chromosomal regions. The PAO1 tetR transposon mutant library is an additional useful resource . Thus both libraries together with any mutant that carries a selective marker can be used to generate easily and rapidly multiple-mutation strains. To allow the construction of multiple mutations in the same chromosome, this method can be used in conjunction with previously described methods for marker excision (Flp/Frt) , which would allow recycling of the same selection marker in the same strain.
The lambda Red-based technique can be used efficiently and rapidly to generate mutants in P. aeruginosa. Chromosomal single gene deletions were generated using PCR product containing flanking regions that are homologous to the target sequence. To maximize the efficiency of mutants' generation, PCR products with 600- to 400-nt homology extension to the target gene should be used. This technique, in conjunction with previously generated P. aeruginosa transposon libraries, can also serve to delete large chromosomal regions.
Strains and growth conditions
Pseudomonas and E. coli strains were grown in Luria Bertani medium (LB) at 37°C with aeration and when necessary pseudomonas cultures were supplemented with kanamycin (400 μg/ml), gentamycin (15 μg/ml) or carbenicillin (300 μg/ml). Pseudomonas strains used in this study include ΔpqsA , ΔpqsE , ΔmvfR , PA14_42880::MAR2xT7 and PA14_43100::MAR2xT7 . The trpE-phnAB- double mutant was constructed in this study: 1293 bp of phnA and 300 bp of phnB were deleted by allelic exchange in the auxotroph trpE::Mar2xT7 mutant background. The vector pKOBEG-sacB  contains the Red operon expressed under the control of the arabinose inducible pBAD promoter and the sacB gene that is necessary to cure the plasmid [2, 4]. Since pKOBEG-sacB was not capable of replication in P. aeruginosa, the Red operon-araC fragment obtained after digestion with Kpn I and Hind III was cloned into the multi clonal site of the E. coli – P. aeruginosa shuttle vector pUCP18 (Genbank U07164)  to create the plasmid pUCP18-Red. In addition, the Nde I sacB fragment from pKOBEG-sacB was cloned into pUCP18-Red, previously digested with Nde I, to create pUCP18-RedS (Genbank EU073163) (Additional file 1). P. aeruginosa recombinants were easily cured from pUCP18-RedS by streaking the mutant strains on NaCl-free LB agar plates supplemented with 10% sucrose.
Approximately 5 μg of DNA from P. aeruginosa and 1 μg of pUC4K DNA was digested by Hind III. The 5' end of the kanamycin cassette was amplified using primers 5'-gccacgttgtgtctcaaaat-3' and 5'-gcatttctttccagacttgttc-3', and the PCR product was labeled using the ECL kit (Amersham) and used for hybridization following manufacturer's instructions.
All PCRs were performed using High Fidelity Taq polymerase (Roche), 1 μM of each primer, 100 μM of dNTPs. DMSO 8% was added to the reaction when P. aeruginosa genomic DNA was used as template. Most of the primers were generated by the Massachusetts General Hospital DNA oligo core facility. The long primers were obtained from Sigma Aldrich.
A kanamycin resistance cassette with long flanking regions homologous to the target gene was generated by a previously described 3-step PCR protocol [4, 20] with minor modifications. P. aeruginosa genomic DNA was first used as a template to amplify the regions flanking the target gene with the primers Fup/Rup-kan and Fdown-kan/Rdown (Additional file 2 and Fig. 1A). Rup-kan and Fdown-kan contain, respectively, at their 5' extremity 19 and 22-nt regions that are homologous to the 5' and the 3' terminals of the kanamycin resistance gene. The plasmid pUC4K  was used as a template to amplify the kanamycin cassette with the primers F-kan/R-kan (Additional file 2). In a 2nd PCR, 10 ng, 25 ng or 50 ng of each of the three fragments were mixed with the primers Fup/Rdown. A third PCR was finally performed using again the Fup/Rdown primers and the product of the second PCR as the template in order to increase the yield of the sought band (Fig. 1A). Five hundred microliters (10 reactions) of the 3rd PCR were ethanol precipitated, dissolved in 10 μl of water and dialyzed for 1 h to eliminate salt that can interfere with electroporation.
The 1-step kynBU or lasR PCR products were generated using pUC4K (Genbank X06404) as template with a mixture of long and short primers (Fig 1B). The long primers contained at their 5' extremity 100-nt homology to the flanking regions of kynBU ( F-kynBU100-kan/R-kunBU100-kan) or lasR (F-lasR100-kan/R-lasR100-kan) and at their 3' extremity 22–24 nt homology to the kanamycin resistance cassette. The short primers F-kynBU/R-kunBU and F-lasR/R-lasR were homologous to the 5' extremities of the kynBU or the lasR long primers, respectively.
The DNA concentration was measured by spectrophotometer at 260 nm and the DNA molecular weight was visually confirmed on agarose gel.
P. aeruginosa PA14/pUCP18-RedS was grown in LB with carbenicillin to an optical density at 600 nm (OD600) of 0.4 at 37°C. Then cells were induced for 1.5 h by treatment with 0.2% L-arabinose; the bacteria were rendered electrocompetent by four washings with 10% sucrose at room temperature and were condensed to a concentration of about 1011 /ml. Electroporation was carried out using 100 μl of bacteria solution and no more than 10 μl of DNA. The electroporated cells were diluted in 2 ml LB and incubated for 2 h at 37°C. Transformants were selected by plating the electroporated cells on antibiotic-imbued plates and further verified by PCR for the correct insertion using two set of primers Fupout/Rinkan and Fupout/Rdownout, with the latter pair hybridizing outside the DNA region used for the recombination event.
- Polymerase Chain Reaction (PCR):
nucleotide (nt), base pair (bp), kan (kanamycin), Kanamycin resistant (KmR).
This work was supported by the National Institute of Health R01AI063433.
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