Transposon sequencing (Tn-seq) experiment

The Tn-seq experiment was performed following the design presented in Fig 1A. Two aliquots of the P. aeruginosa PA14 transposon library previously prepared [89] were thawed on ice for 15 minutes, diluted to a starting optical density (OD₅₀₀) of 0.05 in 50 mL of SMM, and grown aerobically under shaking conditions (250 rpm) at 37°C for approximately 4 to 5 generations to an OD₅₀₀ of 0.8–1. These growing conditions were used for all the stages of the experiment. After growth in SMM, each aliquot was considered an independent replicate. Cells from each replicate were pelleted, washed, and resuspended (5 mL in 18 × 150 mm glass tubes, OD₅₀₀ = 2) in minimal phosphate buffer (MPB; 50 mM KH₂PO₄/K₂HPO₄[pH 7], 42.8 mM NaCl) with and without 100 μM PYO. Cells were then incubated for 26 hours under shaking conditions at 37°C. Therefore, the experiment consisted of 4 different samples that were later sequenced: (i) “R1 No PYO”; (ii) “R1 + PYO”; (iii) “R2 No PYO”; and (iv) “R2 + PYO.” After the incubation, cultures from all treatments were pelleted, washed again to remove PYO, and resuspended in fresh SMM. Immediately, an aliquot of each sample was diluted to a starting OD₅₀₀ of approximately 0.05 in 25 mL SMM, followed by outgrowth for approximately 4 to 5 generations to an OD₅₀₀ of 0.8–1. After outgrowth, 2.5 mL of each sample was pelleted and stored at −80°C.

gDNA was extracted from the pelleted samples using the DNeasy Blood & Tissue kit (Qiagen, Valencia, California, USA). All the steps for sequencing library preparation followed exactly the protocol used by Basta and colleagues [89], including (i) DNA shearing by sonication (to produce 200- to 500-bp fragments); (ii) end repair; (iii) addition of poly(C) tail; and (iv) enrichment of transposon–genome junctions and addition of adapter for Illumina sequencing by PCR [89,90]. The resulting amplified DNA samples were sequenced using 100-bp single-end reads on the Illumina HiSeq 2500 platform at the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech. Data analysis also followed Basta and colleagues [89]. In summary, sequences were mapped to the P. aeruginosa UCBPP-PA14 genome sequence using Bowtie 2 [91] and were analyzed in MATLAB using the ARTIST Tn-seq analysis pipeline [92], with nonoverlapping windows of 100 bp across the genome [89,92]. Using the Mann–Whitney U statistical test, the total reads mapping for each gene in the “+PYO” samples were compared to the corresponding reads in the “No PYO” control for each replicate independently [89,92]. Next, the read ratio for each replicate was calculated within ARTIST for each gene and then log₂ transformed. Finally, the p-values for both replicates were combined using the Fisher combined probability test as done in Basta and colleagues [89], and the average of the log₂ ratios of the 2 replicates are also shown. For the log₂ ratios and p-values for all PA14 genes, see S1 Table. For heatmaps shown in Fig 1A, the average log₂ ratios (fitness) for the selected genes were plotted using the geom_tile() function from the ggplot2 package in R [93,94].