Fig. 2

cydC Is An Essential Gene, and Expression of cydC-D86G is sufficient to confer tolerance to [EMIM]OAc. a E. coli DH1 cells harboring a plasmid-borne copy of either wild type or mutant cydC-D86G [pTE42/P_trc-cydD-cydC or pTE43/P_trc-cydD-cydC-D86G] were untreated or exposed to 75 mM [EMIM]OAc (“Methods”). The change in OD₆₀₀ was measured every fifteen minutes for 40 h. b Evidence that cydC is essential by plasmid shuffle assay. After generating a strain with both plasmid pTE50 and ∆cydC, cells are grown without selection to allow for spontaneous plasmid loss. The rate of plasmid loss in both wild type and ∆cydC strains is compared by selecting for cells without the plasmid on sucrose plates. To allow for spontaneous plasmid loss, cells were grown under non-selective conditions for 24 h at 30 °C. Cells were serially diluted after growth in nonselective media, and plated onto solid LB agar media with or without 10% sucrose. Colonies that are viable in the presence of sucrose have lost the plasmid