Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Lesic
Biliana
B
Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA. lesic@molbio.mgh.harvard.edu
Rahme
Laurence G
LG
D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation; D005818:Genetic Engineering / Q000379:methods*; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011710:Pyocyanine / Q000096:biosynthesis; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D017384:Sequence Deletion
D004269:DNA, Bacterial; D045522:Recombinases; D011710:Pyocyanine