FIG 5.

Influence of nucleotide substitutions in ⁻¹⁰P_farR and PAL1 on expression of FarR, and resistance to linoleic acid. (A) Western blot of gel loaded with 25 ng of purified 6×His-FarR or 25 µg of cell lysate protein from E. coli DH5α transformed with pLI50 vehicle, pLIfarR, or pLIfarER and derivatives containing nucleotide substitutions in ⁻¹⁰P_farR (pLIfarR1 and pLIfarER1), PAL1 (pLIfarER2), or ⁻¹⁰P_farR and PAL1 (pLIfarER3). (B) Luciferase reporter gene assays of S. aureus USA300 transformed with pGYfarR::lux, or pGYfarR1::lux harboring a G→A substitution in ⁻¹⁰P_farR. Triplicates of each culture were grown in TSB, with monitoring of OD₆₀₀ and luciferase activity (relative light units [RLU]) at hourly intervals. Data for RLU determinations are standardized to OD₆₀₀ units (RLU/OD) to account for differences in cell density. (C) Growth of USA300 ΔfarER complemented with pLI50 vehicle or pLIfarER and derivatives in TSB plus 50 µM linoleic acid. Each data point represents the mean and standard deviation of the results from triplicate cultures.