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Functional genomics approaches have shown to be a useful tool in determining the molecular fingerprint which due to the lack of associated functions could alter the toxicity sensitivity to chemicals and hence reveal key pathways associated to toxicity.We hypothesize that the functional gene fingerprint could not only help to understand the mechanisms of toxicity caused by chemicals, but also be used to categorize chemicals.Triclosan (TCS) is a broad-spectrurn biocide with known mode-of-action of lipid synthesis in E.coli.Clarithromycin (CLARY) is a macrolide ribosomal antibiotic which interferes with protein synthesis.Sulfamethoxazole (SMX) is an antibiotic sulphonamide that inhibits the synthesis of dihydrofolic acid which leads to disorder of nucleic acid synthesis.4-Chlorophenol (4-CP), 3,4-Dichloroaniline (DCA), 2, 2, 2-trichloroethanol (TCE) is narcosis chemicals in some species like fish.However, the potential molecular mechanisms using the whole genome library of E.coli knockout mutant are unknown.In this study, molecular mechanisms of three classical known chemicals with different MOA and three candidate narcosis chemicals which were TCS, CLARY,SMX and 4-CP, DCA, TCE were assessed by Escherichia coli (E.coli) genome-wide knockout library that contains 3864 strains of single gene-mutants in a high-throughput manner.A full library screening identified 131,193, 169, 62, 94 and 87 robust hits by TCS, CLARY, SMX, 4-CP,DCA and TCE with two replicates screening, respectively.GO biological pathway analysis on TCS focus on lipid metabolic and biosynthesis process and cellular macromolecular metabolism.The responsive genes of CLARY focus on pathways related to translation, organic substance metabolism and cellular macromolecular metabolism.SMX influenced pathways of nucleotide biosynthesis, phosphorus metabolic process and nitrogen compound metabolic process.The enriched pathways not only validated the known MOA but also explore the other potential adverse effect of TCS, CLARY and SMX.For other three potential narcosis chemicals, 4-CP influenced the GO pathways of lipopolysaccharide biosynthesis, carbohydrate derivative metabolism and lipid metabolism.The pathways of purine-containing compound biosynthesis, phosphorus metabolism, locomotion, response to stimulus, taxis, response to external stimulus were enriched by DCA.The pathways of locomotion, response to stimulus and response to external stimulus also enriched by TCE.It shows that narcosis chemicals by some species may have target in some other species.Responsive gene mutants which were responsive by TCS, CLARY, SMX, 4-CP, DCA and TCE were validated by a full concentration range cytotoxicity test, respectively.The most responsive genes by each chemical were selected for further chemical clustering study.90 responsive gene mutants were used to cluster 30 more other chemicals with known mode-of-action.We hypothesis that the fitness sensitivity fingerprint of chemicals by these responsive genes would distinguished (1) narcosis chemicals and non-narcosis chemicals;(2) different specific mode-of-action chemicals.