Sezer Kürkçü Merve, Onat Taşdelen Kadriye Aslıhan, Öztürkel Kabakaş Hatice, Dibek Esra, Babayeva Anara, Elgin Emine Sonay, Çöl Bekir
Research and Application Center for Research Laboratories (ALM Research Building), Mugla Sitki Kocman University, Mugla, 48000, Türkiye.
Biotechnology Research Center (ALM Research Building), Mugla Sıtkı Koçman University, Mugla, 48000, Türkiye.
World J Microbiol Biotechnol. 2025 Aug 9;41(8):307. doi: 10.1007/s11274-025-04506-4.
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., ∆aaeX, ∆aaeA, ∆seqA, ∆vacJ, ∆dksA) displayed growth inhibition, while resistant mutants (e.g., ∆yhfK, ∆hofQ, ∆ybaT, ∆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., ∆dksA, ∆seqA, ∆aaeA, ∆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.
反式肉桂酸（tCA）是一种具有抗菌活性的天然酚类化合物，其调控细菌敏感性和抗性的分子机制尚不清楚。本研究利用大肠杆菌K-12 Keio单基因敲除文库（3985个突变体）进行全基因组筛选，以鉴定调节tCA反应的基因。在补充有tCA（0-1.5mg/mL）的LB培养基上筛选突变体。通过视觉和定量评估验证的表型分析确定了78个敏感突变体和52个抗性突变体。在tCA胁迫下的连续点样试验证实了分类：敏感突变体（如∆aaeX、∆aaeA、∆seqA、∆vacJ、∆dksA）表现出生长抑制，而抗性突变体（如∆yhfK、∆hofQ、∆ybaT、∆groL）表现出生长增强。利用来自SGA Tools、全基因组筛选（GWS）和点样试验的综合数据，将突变体分为高、中、低敏感性/抗性组，产生了18个高敏感（HS）、43个中敏感（MS）、17个低敏感（LS）、20个高抗性（HR）、23个中抗性（MR）和9个低抗性（LR）菌株。通过质粒用野生型等位基因对敏感突变体（如∆dksA、∆seqA、∆aaeA、∆vacJ）进行互补恢复了生长并提高了最低抑菌浓度（MIC），直接将这些基因与tCA敏感性联系起来。功能评估（EcoCyc、DAVID、STRING）显示，HS基因命中与转录调控、代谢活性、蛋白质折叠、DNA修复、转运和膜稳定性相关。抗性基因命中与应激反应和解毒途径相关。这种系统水平的分析阐明了大肠杆菌对tCA反应的遗传基础，确定了利用tCA或其衍生物的抗菌策略的靶点。
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