Fig. 3: A transposon mutant library screen uncovers genetic determinants of biofilm stimulation by sub-MIC antibiotics.
a A PAO1 transposon library was constructed and arrayed in 96-well format, then screened for biofilm stimulation by ½ MIC cefixime in technical duplicate. Non-stimulated mutants were identified as having an average cefixime-induced biofilm of less than 200% of the vehicle control. Hits were validated in dose-response peg lid assays using cefixime, tobramycin, and thiostrepton and those lacking biofilm stimulation in response all three compounds were selected for follow up. The location of transposon insertions was identified by arbitrarily primed touchdown PCR followed by sequencing. b A replicate plot showing biofilm stimulation by ½ MIC cefixime for the entire transposon library (~13 000 mutants) across both technical replicates. The axes are shown as a log10 scale, and the dashed line shows the 200% of untreated control cutoff for hits. Each mutant is represented by a circle, with yellow circles showing the hits that failed to respond to three different antibiotics in dose-response follow-up assays. The identity of each verified hit is shown in the inset box. c A schematic of the genes that, when disrupted, prevent biofilm formation in response to antibiotics is shown, with the genes of interest in different colors and neighboring genes in gray. The predicted gene product description according to the Pseudomonas database79 for each hit is on the left. Source data are provided in Supplementary Data Set 1.
