Comparative analysis of genome-wide footprinting data suggests that transposon insertions in or near 586 genes (see Dataset S3 for a complete list of these genes) cause reproducible, condition-dependent behavior that increases fitness in at least one setting (see Protocol S1). (A) The 586 genes (rows) were arranged using K-means clustering into six clusters shown on the left (C1 through C6). The hybridization scores shown for each gene were mean-centered and normalized to a standard deviation of one. This commonly used normalization puts each gene's fitness profile on a similar scale and facilitates comparison between the different conditions. Yellow indicates those conditions where mutants with transposons in or near the indicated gene underwent the greatest increases in abundance. Blue indicates conditions where transposons in or near the same gene were either deleterious or were slightly beneficial and resulted in a comparatively small increase in abundance. Column labels indicate the experimental condition: Bio-ND and Bio-TOB refer to transposon insertion libraries grown as biofilms and treated with no drug or tobramycin, respectively, and Pla-ND and Pla-TOB refer to libraries grown planktonically without or with tobramycin. Two biological replicates were performed in each condition and numbers indicate the repetition number. Gene names and annotations are in Dataset S3. (B) iPAGE was used to look for enrichment and depletion of functional categories (rows) among clusters C1 through C6 plus the set of genes not in any cluster (columns). Red (green) indicates that genes in the cluster were enriched (depleted) for the indicated category.

(A) The NADH/NAD⁺ ratio was measured for a subset of mutants with the most pronounced growth advantage in the planktonic state with tobramycin. The NADH/NAD+ ratios for the strains shown are significantly higher than wild-type (Student's one-sided t-test p-values: 0.02, 0.002, 0.001, and 0.015 for mutants in PA1329, PA3966, PA5207, and nuoK, respectively). Error bars represent the standard error of at least three replicates. (B) The disruptive effect of tobramycin on the outer membrane of different mutants was measured using an NPN assay. NPN and tobramycin were added at the indicated times.

Shown are pathways that the genes from Figure 3A and 3B likely use to modulate biofilm-mediated antibiotic tolerance in P. aeruginosa. The “*” indicates that the altered antibiotic-susceptibility is specific to the biofilm state. Genes in the ‘Other' category likely affect uncharacterized pathways or pathways not assayed in this work.

For biofilm experiments, a transposon insertion library was given 24 hours to form a biofilm on a plastic slide in media lacking tobramycin. Next, the slide and the attached biofilm were moved to fresh media with tobramycin for another 24 hours, and then the biofilm was allowed to recover in fresh, drug-free media for an additional 24 hours. After repeating the drug exposure and recovery a second time, the biofilm was disrupted and the cells were collected. Abundance of individual mutants was determined using microarray-based genetic footprinting. Planktonic experiments were similar except the slide was not included and cultures were shaken. Tobramycin was omitted from “no drug” controls. In all cases, containers were sealed. See Materials and Methods for details.

(A, B) Competitions were started with equal amounts of mutant and reference cells. The y-axis indicates the relative count of mutants over the reference strain following one round of the indicated experimental challenge (as explained in Materials and Methods). As cultures undergo different numbers of generations during each type of challenge, values from different challenges for the same mutant are not directly comparable. Error bars indicate the standard error of at least 8, 4, and 3 experiments for the Bio-TOB, Bio-ND, and Pla-ND conditions, respectively. Mutants in (A) have an advantage over wild-type in the Bio-TOB competition; mutants in (B) have a disadvantage. Gene annotations, which were updated from the original genome annotation [48] by BLAST comparisons [14] against the NCBI non-redundant database, are in Table S3. (C) Growth curves for representative strains from panels (A) and (B) with 4 µg/ml tobramycin are shown.

(A) The MPAO1 strain with either an empty vector or a plasmid containing PA0614 under the control of an arabinose-inducible promoter was grown in M63 medium in the presence and absence of 0.2% arabinose (Ara). (B) A gfp promoter fusion was used to measure the expression of PA0614 in both biofilm and planktonic settings, in the presence and absence of 8 µg/ml tobramycin. Promoter activities are normalized by colony forming units (CFU). (C) Expression differences between exponentially growing cultures of wild-type and PA3726 mutant cells were sorted and partitioned into 20 equally populated bins, which were subjected to iPAGE analysis. The most informative functional categories are shown. (D) PA3726 promoter activity was measured as described in (B).