General scheme of construction of suicide vectors (A) and replacement of target genes (B).

A: For construction of suicide vectors for replacement of target genes, regions of approximately 400 bp flanking the target up- and downstream, respectively, were amplified using primers with integrated restriction sites allowing directed insertion into mobilisable vector pEX18Ap. PCR-products of upstream regions (1) were cleaved with EcoRI and BglII and downstream regions (2) with HindIII and BglII (only in case of PA1793 BamHI instead of BglII). The gentamycin-GFP cassette (3) of pPS858 was excised using BamHI and vector pEX18Ap was linearised with EcoRI and HindIII. Cleaved fragments 1, 2, and 3 and linearised vector pEX18Ap (4) were then combined in ligation mixture. The gentamycin-GFP cassette ligation to up and downstream fragments aided by the compatible cohesive ends between BamHI and BglII. Then E. coli donor strain ST18 was transformed with this mixture. Afterwards the suicide constructs were transferred from E. coli ST18 into P.aeruginosa PAO1 by conjugation. B: After conjugational transfer of suicide constructs recombinational replacement of the target genes and loss of vector backbone was forced by sucrose counter selection and gentamycin selection, respectively. Total DNA was isolated from knock out mutants and correct gene replacement was confirmed by sequencing extended PCR-products of the flanking regions.