Genome-wide screening of the Keio collection identifies genetic determinants of Escherichia coli sensitivity and tolerance to benzoic acid.

Benzoic acid (BA) is a widely used weak-acid preservative, yet the genome-scale mechanisms underlying bacterial adaptation to preservative stress in food-associated environments remain incompletely defined. In this study, we screened the Escherichia coli Keio single-gene mutant line to identify genes associated with altered BA sensitivity or tolerance. Mutants were evaluated on LB agar containing 0.70-1.00 mg/mL BA, and growth phenotypes were assessed across multiple incubation times using visual scoring and a Growth Inhibition Index (GII). Putative hits were further examined by sequential spot assays, and selected sensitive mutants were subjected to complementation analysis using ASKA clones carried on pCA24N. Genome-wide screening identified 44 BA-sensitive and 153 BA-tolerant mutants with available annotations. Sensitive mutants were associated mainly with membrane-linked transport, phospholipid homeostasis, ion transport, and redox-related functions, whereas tolerant mutants were linked predominantly to respiratory processes, carbon metabolism, regulatory pathways, and stress adaptation. Phenotypic validation supported the screening results, and complementation restored or improved growth in several mutants, including Δppk, ΔpstA, ΔycdO, ΔyrbF, ΔmodB, and ΔrrmJ. Overall, these findings show that the E. coli response to BA is shaped by interconnected processes involving membrane integrity, transport systems, energy metabolism, and regulatory adaptation. These findings provide a systems-level framework for understanding bacterial adaptation to benzoic acid and may support future strategies aimed at improving preservative efficacy and microbial control in food systems.