FIG. 5.

Depiction of the two-step procedure used to transfer algB alleles to the P. aeruginosa chromosome by allelic exchange. (A) Generation of ΔalgB::ΩaacC1 intermediate strain FRD840. Plasmid pUS65 (pEX100T plus ΔalgB::ΩaacC1), which cannot replicate in P. aeruginosa, was mobilized into strain FRD1 and single-crossover recombinants were isolated by selection for Gm^r. Most Gm^r bacteria were also Cb^r, indicating integration of the entire plasmid (single-crossover events). Double recombinants were isolated by plating Gm^r bacteria on agar containing 5% sucrose. The desired (double) recombinants were Gm^r Cb^s (marker on pUS65). (B) Allele replacements. To perform allele replacements, pEX100T containing a specific algB allele (algB45 in this example) was mobilized into the intermediate strain FRD840, and single recombinants were selected by resistance to carbenicillin. The desired allele replacements (Gm^s Cb^s) were then obtained by counterselection on sucrose-containing medium. Abbreviations: ori, ColE1 origin; sacB, gene encoding levansucrase; oriT, transfer origin; bla, resistance to carbenicillin; aacC1, resistance to gentamicin; s, antibiotic sensitivity; Cb, carbenicillin; Gm, gentamicin; H, HindIII; K, KpnI; R, EcoRI; X, XhoI.