Characterization of the Pf4 transfer; (A) Pf4 transfer was observed among PAO1 cells; PAO1 cells labeled with tetracycline (Tet^R) and gentamicin (Gen^R) resistance carry Tet^R genes in the Pf4 locus and the pPSV37 plasmid, respectively; the same labeling applies to the rest of the figures unless otherwise specified; 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; (B) fluorescence microscopy of isolated PAO1 daughter cells; the parental PAO1 strains included phoA::mRFP Pf4_Tet^R as the donor and phoA::sfGFP carrying pPSV37 plasmid as the recipient; the daughter cells were collected from the overnight LB-agar plates with or without antibiotic selection as indicated, and incubated in LB with 1 mM IPTG for 30 min prior to imaging; cells were placed on agarose pads (~0.13 mm thick) for imaging to restrict their movement; a 30 × 30 μm representative field of cells is shown; scale bar, 5 μm; (C) Pf4 transfer among PAO1 daughter cells; top: schematic showing isolated G1+ and G1− daughter cells; middle: Pf4 transfer assay between PAO1 Pf4_Carb^R strains with initial PAO1 Gen^R, G1+ or G1− daughter cells, respectively; bottom: quantification of the Pf4 transfer; the transfer fraction was calculated as the ratio of daughter cells with dual resistance to carbenicillin and gentamicin to all cells with gentamicin resistance in each sample; error bars indicate the standard deviation of five biological replicates, and statistical significance was calculated using one-way ANOVA (analysis of variance) test; ns, not significant; ND, not detected; (D) Pf4 transfer assay between different PAO1 mutants; the plasmid-borne excisionase XisF4 (pXisF4) was used to stimulate Pf4 production; for (A), (C), and (D), PAO1 ∆PA0717–PA0726 mutants were used as the ∆Pf4 control.