Escherichia coli and P. aeruginosa strains were maintained on Luria-Bertani medium (LB; 10 g per liter tryptone, 5 g per liter yeast extract, 10 g per liter NaCl; Becton, Dickinson & Co., Sparks, MD). For plasmid maintenance in E. coli, the medium was supplemented with 100 μg per ml ampicillin, 25 μg per ml chloramphenicol, 35 μg per ml kanamycin or 15 μg per ml gentamycin. For marker selection in P. aeruginosa, 200 μg per ml carbenicillin and 30 μg per ml of gentamycin was used, as appropriate.
β-galactosidase activity assays
Cells were grown at 37°C with shaking to exponential phase (optical density at 540 nm ~0.4–0.8) in LB medium with 0.05% Brij 58 +/- 0.05% oleic acid. β-galactosidase activity was measured in chloroform/sodium dodecyl sulfate-permeabilized cells and its activity calculated as previously described .
Standard DNA procedures
Routine procedures were employed for manipulation of DNA . Plasmid DNAs were isolated using the QIAprep Mini-spin kit (Qiagen, Valencia, CA) and P. aeruginosa chromosomal DNA was obtained using the QIAamp DNA Mini Kit. DNA fragments were purified from agarose gels utilizing the QIAquick gel extraction kit.
Construction of the Gateway-compatible gene replacement vector pEX18ApGW
The previously described gene replacement vector pEX18Ap  was modified by cloning of a 1,755 bp Hin dIII-Kpn I fragment from pUC18-mini-Tn7 T-Gm-GW (GenBank accession number AY737004), followed by transformation into the gyrA strain DB3.1 (Invitrogen).
1st round PCRs
PCR-amplification of the gentamycin resistance gene cassette
A 50 μl PCR reaction contained 5 ng pPS856  template DNA, 1x HiFi Platinum Taq buffer, 2 mM MgSO4, 200 μM dNTPs, 0.2 μM of Gm-F and Gm-R (Table 1) and 5 units of HiFi Platinum Taq polymerase (Invitrogen). Cycle conditions were 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 1 min 30 s, and a final extension at 68°C for 7 min. The resulting 1,053 bp PCR product was purified by agarose gel electrophoresis and its concentration determined spectrophotometrically (A260 nm) in an Eppendorf Biophotometer (Hamburg, Germany) and using the 50–2000 μl Eppendorf UVettes
PCR-amplification of 5' and 3' gene fragments
Two 50 μl PCR reactions were prepared. The first reaction contained 20 ng chromosomal template DNA, 1x HiFi Platinum Taq buffer, 2 mM MgSO4, 5% DMSO, 200 μM dNTPs, 0.8 μM of PA1520-UpF-GWL and PA1520-UpR-Gm (Table 1) and 5 units of Platinum Taq polymerase. The second reaction contained the same components as the first, but 0.8 μM of PA1520-DnF-Gm and PA1520-DnR-GWR (Table 1). Cycle conditions were 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 30 s, and a final extension at 68°C for 10 min. The resulting PCR products were purified by agarose gel electrophoresis and their concentrations determined spectrophotometrically (A260 nm).
2nd round PCR
A 50 μl PCR reaction contained 50 ng each of the PA1520 5' and 3' purified template DNAs, and 50 ng of FRT-Gm-FRT template DNA prepared during 1st round PCR. The reaction mix also contained 1x HiFi Platinum Taq buffer, 2 mM MgSO4, 5% DMSO and 200 μM dNTPs, and 5 units of HiFi Platinum Taq polymerase. After an initial denaturation at 94°C for 2 min, 3 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min were run without added primers. The third cycle was paused at 30 s of the 68°C extension, primers GW-attB1 and GW-attB2 were added to 0.2 μM each, and the cycle was then finished by another 30 s extension at 68°C. The PCR was completed by 25 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 5 min, and a final extension at 68°C for 10 min. The resulting major PCR product was purified by agarose gel electrophoresis and its concentrations determined spectrophotometrically (A260 nm). The identity of the PCR fragment was confirmed by Xba I digestion (each FRT site of the FRT-Gm-FRT fragment contains a Xba I site).
BP and LR clonase reactions
The BP and LR clonase reactions for recombinational transfer of the PCR product into pDONR221 and pEX18ApGW, respectively, were performed as described in Invitrogen's Gateway cloning manual, but using only half of the recommended amounts of BP and LR clonase mixes and either E. coli DH5α  or HPS1  as host strains. The presence of the correct fragments in transformants obtained with DNA from either clonase reaction was verified by digestion with Xba I because each FRT site flanking the Gmr gene contains a Xba I site. However, before plasmid isolation from transformants obtained with DNA from the LR clonase reaction, 25–50 transformants were i) patched on LB+Km and LB+Ap plates, and ii) simultaneously purified for single colonies on LB+Ap plates. This was necessary to distinguish between those colonies containing only the desired pEX18ApGW-Gene::Gm from those containing this plasmid and the frequently contaminating pDONR-Gene::Gm (pEX18Ap-derived plasmids confer Apr and pDONR plasmids confer Kmr). Plasmids were then isolated from Apr Kms transformants and analyzed by Xba I digestion. In those extremely rare instances where all patched transformants contained a mixed plasmid population (<1% of to date performed LR clonase reactions), retransformation with Apr selection was necessary to obtain colonies containing only pEX18ApGW-Gene::Gm.
Transfer of plasmid-borne deletions to the P. aeruginosa chromosome
A rapid electroporation method described elsewhere  was used to transfer the pEX18ApGW-borne deletion mutations to P. aeruginosa. Briefly, 6 ml of an overnight culture grown in LB medium was harvested in 4 microcentrifuge tubes by centrifugation (1–2 min, 16,000 × g) at room temperature. Each cell pellet was washed twice with 1 ml of room temperature 300 mM sucrose and they were then combined in a total of 100 μl 300 mM sucrose. For electroporation, 300-500 ng of plasmid DNA was mixed with 100 μl of electrocompetent cells and transferred to a 2 mm gap width electroporation cuvette. After applying a pulse (settings: 25 μF; 200 Ohm; 2.5 kV on a Bio-Rad GenePulserXcell™), 1 ml of LB medium was added at once, and the cells were transferred to a 17 × 100 mm glass or polystyrene tube and shaken for 1 h at 37°C. The cells were then harvested in a microcentrifuge tube. 800 μl of the supernatant was discarded and the cell pellet resuspended in the residual medium. The entire mixture was then plated on two LB plates containing 30 μg per ml Gm (LB+Gm30). The plates were incubated at 37°C until colonies appeared (usually within 24 h). Under these conditions, the transformation efficiencies were generally 30–100 transformants per μg of DNA. A few colonies were patched on LB+Gm30 plates and LB+Cb200 plates to differentiate single- from double cross-over events. To ascertain resolution of merodiploids, Gmr colonies were struck for single colonies on LB+Gm30 plates containing 5% sucrose. Gmr colonies from the LB-Gm-sucrose plates were patched onto LB+Gm30+5% sucrose, as well as LB plates with 200 μg per ml carbenicillin (LB+Cb200). Colonies growing on the LB-Gm-sucrose, but not on the LB-carbenicillin plates were considered putative deletion mutants. The presence of the correct mutations was verified by colony PCR. To do this, a single, large colony (or the equivalent from a cell patch) was picked from a LB-Gm-sucrose plate, transferred to 30 μl H2O in a microcentrifuge tube and boiled for 5 min. Cell debris was removed by centrifugation in a microcentrifuge fuge (2 min; 16,000 × g), and the supernatant was transferred to a fresh tube which was placed on ice. 5 μl of the supernatant was used as source of template DNA in a 50 μl PCR reaction containing Taq buffer, 1.5 mM MgSO4, 5% DMSO, 0.6 μM each of PA1520-UpF-GWL and PA1520-DnR-GWR, 200 μM dNTPs and 5 units Taq polymerase (Fermentas, Hanover, MD). Cycle conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 45 s, 55°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 10 min. PCR products were analyzed by agarose gel electrophoresis.
Flp-mediated marker excision
Electrocompetent cells of the newly constructed mutant strain were prepared as described in the preceding paragraph and transformed with 20 ng of pFLP2  DNA as described above. After phenotypic expression at 37°C for 1 h, the cell suspension was diluted 1:1,000 and 1:10,000 with either LB or 0.9% NaCl, and 50 μl aliquots were plated on LB+Cb200 plates and incubated at 37°C until colonies appeared. Transformants were purified for single colonies on LB+Cb200 plates. Ten single colonies were tested for antibiotic-susceptibility on LB ± Gm30 plates and on a LB+Cb200 plate. Two Gms Cbr isolates were struck for single colonies onto a LB+5% sucrose plate and incubated at 37°C until sucrose-resistant colonies appeared. Ten sucrose-resistant colonies were retested on a LB+5% sucrose (master) plate and a LB+Cb200 plate. Finally, two sucrose-resistant and Cbs colonies were struck on LB plates without antibiotics, and their Cbs and Gms phenotypes confirmed by patching on LB ± Cb200 and LB ± Gm30 plates. Deletion of the Gmr marker was assessed by colony PCR utilizing the conditions and primers described in the preceding paragraph