Figure 2
Positive control system.
(a) Strains OFLRΔlacIΔlacZ + PlacZ-gfp and lacIqΔlacZ + PlacZ-gfp have survival fractions (persister frequencies) of 0.46 ± 0.05 and 0.05 ± 0.02, respectively, after 5 h treatment with OFL (5 μg/mL) in stationary phase. Data are averages of 3 biological replicates; error bars portray standard error of the mean (s.e.m.). (b) Histograms of strains OFLRΔlacIΔlacZ + PlacZ-gfp and lacIqΔlacZ + PlacZ-gfp. Histograms are representative of 3 biological replicates. (c) Histogram of a 90% lacIqΔlacZ + PlacZ-gfp and 10% OFLRΔlacIΔlacZ + PlacZ-gfp mixture. A-B-C-D designates FACS quantiles used for OFL tolerance assays shown in (d,e). Quantiles C and D both contain 25% of the population. Quantile A contains either 25% or the smallest possible fraction of the population based on the sorter’s ability to resolve low-fluorescing events and quantile B contains 25% or the remaining fraction of the population. Histogram is representative of 3 biological replicates. (d) NPPs of FACS-sorted samples. Expected NPPs were calculated based on survival fractions (persister frequencies) of the individual strains after 5 h OFL (5 μg/mL) treatment (a) and the knowledge that OFLRΔlacIΔlacZ + PlacZ-gfp falls in quantile D. An approximate 4-fold increase in survival in the HFQ (quantile D) as compared to the LFQs (quantiles A, B, C) was expected. When 2 million cells were sorted from the total population using FACS, a statistically-significant ~4-fold increase in survival was seen in the HFQ as compared to the LFQs (t-test, p-value ≤ 0.05), confirming the expected results. Results are averages of 3 biological replicates; error bars portray s.e.m. (e) Positive control system has high NPP in HFQ (quantile D) as compared to LFQs (quantiles A, B, C). Two million and 200,000 were sorted from a 90/10 lacIq/OFLR mixture. 800,000 and 80,000 were sorted from a 90/10 lacIq/OFLR2 mixture. Data are averages of 3 biological replicates; error bars portray s.e.m.
