Functional dissection of the genome of <i>Salmonella</i> Typhimurium to understand its tolerance to the bactericidal activity of peracetic acid

A genome-saturated Tn5 mutant library (input pool) was inoculated in two replicates into either 6% chicken meat extract (CME) or 11% diluted Luria-Bertani (LB) broth, both supplemented with 15 ppm PAA. Cultures were incubated for 90 minutes at 37°C. Viable Tn5 mutant cells recovered on LB agar plates were combined to form four output pools (two CME and two LB). Genomic DNA extracted from these pools were deep sequenced (Tn5-junction reads). Conditionally essential genes required for fitness in 6% CME and 11% LB were identified and subjected to pathway enrichment analysis (ShinyGO graphical gene-set enrichment tool). We identified two overlapping sets of conditionally essential genes (276 common genes) required for survival in the presence of PAA. In CME, 362 conditionally essential genes were identified, while LB media revealed 536 genes. Pathway enrichment analysis showed that these genes were significantly enriched in pathways such as pyruvate metabolism, the tricarboxylic acid cycle, fumarate reductase/succinate dehydrogenase (transmembrane subunit and 2Fe-2S iron-sulfur cluster binding domain), stress response, and oxidoreductase activity. Notably, genes previously shown to increase sensitivity to PAA upon inactivation (sdhC, zwf, pta, and icdA) were identified as conditionally essential in this study, further validating the Tn-seq data.