Genome-Wide Screen using <i>Escherichia coli</i> Keio knockout mutant line reveals genes related to the antimicrobial properties of trans-Cinnamic Acid

Trans-cinnamic acid (tCA), a naturally occurring phenolic compound with antimicrobial activity, has poorly understood molecular mechanisms governing bacterial sensitivity and resistance. This study employed a genome-wide screen of Escherichia coli K-12 Keio single-gene knockout library (3,985 mutants) to identify genes modulating tCA response. Mutants were screened on LB medium supplemented with tCA (0-1.5 mg/mL). Phenotypic analysis identified 78 sensitive and 52 resistant mutants, validated through visual and quantitative assessments. Sequential spot assays under tCA stress confirmed classifications: sensitive mutants (e.g., triangle aaeX, triangle aaeA, triangle seqA, triangle vacJ, triangle dksA) displayed growth inhibition, while resistant mutants (e.g., triangle yhfK, triangle hofQ, triangle ybaT, triangle groL) exhibited enhanced growth. Mutants were categorized into High, Moderate, or Low Sensitivity/Resistance groups using integrated data from SGA Tools, genome-wide screening (GWS), and spot testing, yielding 18 High Sensitive (HS), 43 Moderate Sensitive (MS), 17 Low Sensitive (LS), 20 High Resistant (HR), 23 Moderate Resistant (MR), and 9 Low Resistant (LR) strains. Complementation of sensitive mutants (e.g., triangle dksA, triangle seqA, triangle aaeA, triangle vacJ) with wild-type alleles via plasmids restored growth and elevated minimum inhibitory concentrations (MICs), directly linking these genes to tCA sensitivity. Functional evaluation (EcoCyc, DAVID, STRING) revealed HS gene hits were associated with transcriptional regulation, metabolic activity, protein folding, DNA repair, transport, and membrane stability. Resistant gene hits were linked to stress response and detoxification pathways. This systems-level analysis elucidates the genetic basis of E. coli's response to tCA, identifying targets for antimicrobial strategies leveraging tCA or its derivatives.