Figure 1. A dual-fluorescence reporter cassette for real-time tracking of adaptive mutations of different types.

(A) Reporter cassette construct for chromosomal insertion. p₀ = 188 bp random DNA sequence, RBS = ribosomal binding site, hairpins = transcriptional terminators, tetA-yfp = selected gene, cfp = constitutively expressed amplification reporter. A, B, C, D = intergenic chromosomal insertion loci, oriC = origin of replication. (B) Immediate chromosomal neighborhoods of loci A-D. Black arrows = essential genes. White arrows = non-essential genes. Grey arrows = no essentiality data available. Patterned arrow (yoeD) = pseudogene. Orange = cryptic prophage CP4-44. Green = origin of replication (oriC). Chromosomal neighborhoods of loci B, C, and D are shown reversed with respect to conventional chromosome coordinates, so that the orientation relative to the reporter cassette is shown in the same way for all four loci. Reporter cassette genes are not drawn to scale. (C) Example fluorescence trajectories of rescued populations with YFP or YFP+CFP (amplification) fluorescence phenotype. RFU = relative fluorescence units (see Methods), yellow and blue lines = YFP and CFP fluorescence, dotted lines = threshold for phenotype classification. (D) Increase of tetracycline concentration in ten-day experiment, normalized to strain-specific minimal inhibitory concentration (MIC, dotted line). (E) qPCR validation of CFP fluorescence as an indicator of extent of amplifications. x-axis: tetA-yfp copy number as determined by qPCR on genomic DNA of rescued population with a YFP+CFP fluorescence phenotype. Error bars = SD of technical qPCR triplicates. r is the Pearson correlation coefficient and P its p-value. RFU = relative fluorescence units, line = linear fit.

DOI: http://dx.doi.org/10.7554/eLife.25100.002

Figure 1—figure supplement 1. Fine-scale determination of MICs of tetracycline for ancestor strains used in experimental evolution.

OD₆₀₀ (platereader units) after 24 hr is shown across tetracycline concentrations (triplicates). Panel columns = integration loci of the reporter cassette, panel rows = genetic background. Note the different scaling of the x-axis for D strains. We define MIC (dashed vertical lines and inset values) as the lowest concentration that restricts growth to OD₆₀₀ ≤0.075 (= OD_t, plate reader units, dashed horizontal lines) in all three replicates. We regard the highest replicate value of strain E at 2 µg/mL as an outlier uninformative about ancestral drug sensitivity, as this culture showed highly increased CFP fluorescence indicative of reporter cassette amplification. The selective conditions in evolution experiments (i.e., tetracycline concentrations) were adjusted according to strain-specific MICs to make results more comparable between strains. Without such an adjustment in tetracycline concentrations, different MICs would cause large differences in population sizes and consequently in the probability of acquiring beneficial mutations.

DOI: http://dx.doi.org/10.7554/eLife.25100.005