Fig. 1.

Enrichment, identification, and characterization of a mutant tolerant to killing by phenol. (A) Survival of wild-type E. coli after multiple rounds of challenge with 3.5 mg/mL phenol. (B) Reduced rate of killing from 3.5 mg/mL phenol by a phenol-tolerant mutant (Phen-T), obtained from A. (C) Elevated concentrations of phenol required to kill the Phen-T mutant during a 15-min treatment. (D) PtsI defect is responsible for phenol tolerance. E. coli cultures were treated with 3.5 mg/mL phenol prior to measurement of survival. Strains: wild-type, Phen-T mutant, ΔptsI mutant, ptsI point (I393S) mutant, Phen-T mutant back-crossed with wild-type ptsI. (E) Expression of ptsHI operon from a plasmid complemented the Phen-T tolerance phenotype. Killing measurements (as in B) were used with the following strains: wild-type, wild-type harboring pACYC184 vector plasmid, Phen-T, Phen-T harboring pACYC184, and Phen-T harboring pACYC184-ptsHI (expressing the ptsHI operon from the plasmid). (F) Mutations in dgcM failed to account for phenol tolerance of the Phen-T mutant. Cultures of wild-type cells and Phen-T, ΔdgcM::Kan, and dgcM G228A mutants were treated and processed as in B. (G and H) Phen-T and ptsI deficiencies have little effect on culture growth. Overnight cultures of wild-type cells and the Phen-T and the ΔptsI mutants were diluted 2,000-fold into fresh LB medium and grown aerobically at 37 °C; at the indicated times turbidity (OD₆₀₀, G) or colony-forming units (H) were determined. Data represent average of three biological replicates; error bars indicate SEM. See SI Appendix, Tables S1 and S2 for supporting information.