Pf4 transfer occurs under multiple conditions; (A) Pf4 transfer assay between different PAO1 populations in a multispecies environment; the donor strains of PAO1 mixtures are indicated, while the recipients were PAO1 strains carrying pPSV37; PAO1 mixtures were co-incubated with or without other species as specified; (B) Pf4 transfer assay between different PAO1 populations using liquid LB medium or ASM as indicated; (C) Pf4 transfer assay using PAO1 wild type (WT) strains or ∆retS mutants as recipients; (D) Pf4 transfer assay between different PAO1 populations using the mouse lung infection model; (E) Pf4 transfer assay between PAO1 Pf4_Tet^R mutants and P. aeruginosa clinical isolates with streptomycin-resistant (Str^R); (F) Pf5 transfer assay between PA14 strains; PA14 cells were labeled with Tet^R inserted in the Pf5 locus and Gen^R conferred by the pPSV37 plasmid, respectively; ∆Pf5, the PA14 ∆EIP97_20555-EIP97_20600 mutant; for (D) and (F), the transfer fraction was calculated as the ratio of daughter cells with dual resistance to tetracycline and gentamicin to all cells with gentamicin resistance in each sample; Error bars indicate the standard deviation of three biological replicates; ND, not detected; For (A) to (E), PAO1 ∆PA0717–PA0726 mutants were used as ∆Pf4 controls.