Complementation of mutants.

To complement the knockout mutant of PA0335, the multicopy-number E. coli-P. aeruginosa shuttle vector pAK1900 was used (47). Gene PA0335 with its promoter region was PCR amplified using the forward primer pAK-PA0335-S and the reverse primer pAK-PA0335-A with restriction sites. The digested PCR product was ligated to pAK1900 to produce the plasmid pAK-0335. pAK-0335 was then transformed into PAO1(Δ0335) by electroporation. Integrants were selected on PIA plates containing 250 μg/ml Cb. The resulting strain was designated PAO1(Δ0335)C. PAO1(Δ0334-5)C1, PAO1(Δ0334-5)C2, and PAO1(Δ0334-5)C3, PAO1(Δ0335)C, and PAO1(Δ0335)C1 were constructed similarly.