1. Complex Interplay of Physiology and Selection in the Emergence of Antibiotic Resistance 
Wei-Hsiang Lin, Edo Kussell 
Current Biology 
Volume 26, Issue 11, Pages (June 2016)
DOI: /j.cub
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2. Figure 1
Stochastic Expression of tetA and Population Dynamics in a Fluctuating Environment
(A) The stochastic switch uses chromosomal agn43 promoter driving T7 RNAP [19], which activates tetA and GFP expression from plasmids.
(B) Heterogeneity in agn43 promoter expression. All cells constitutively express mCherry, while the agn43 promoter state is reported by GFP. Scale bar, 5 μm.
(C) Upper panel shows trajectories of six different populations for period duration T = 3 hr. Tetracycline (Tc) killing phases are indicated by gray shades. Growth rate is measured by the optical flow (see Supplemental Experimental Procedures); growth rate in non-selective Luria-Bertani (LB) medium is indicated by the horizontal line. Lower panel shows dynamics of resistance sweeps tracked by the frequency of ON cells in each population.
(D) Progression of resistance sweeps across all populations of the selectable (Sel.) strain for each period duration T shown in different colors. The limit of extremely short durations T corresponds to a constant, “average” environment with 3 μg/ml Tc (Avg Env).
(E) Progression of antibiotic killing for different period durations T using the control (Ctl.) strain; see legend for (D) for period duration colors.
See also Figure S1.
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3. Figure 2
Long-Term Growth Rates, ON Cell Frequency, and Efficacy of Selection
(A) Correlation between ON cell frequency (x axis) and population long-term growth rate (y axis) for different T. Each point represents a population. Red and blue dots represent data averaged over time intervals 0–12 hr and 24–36 hr, respectively, from experiments using the Sel. strain. Purple dashed lines, growth rate in non-selective LB medium.
(B) Effective growth rates Gon and Goff for ON (green) and OFF (black) cells at different T obtained by linear regression of ON cell frequency versus growth rate; error bars correspond to the SD estimated from the regression model. Purple dashed line, growth rate in non-selective LB medium. Single-cell measurement yields similar results (Figure S1D).
(C) Selective coefficient of ON versus OFF cells for Sel. and Ctl. strains, using s = (Gon – Goff) / Goff. Values for Sel. indicate the advantage of resistant ON cells. Ctl. strains go extinct under fast fluctuations; hence, no data are shown for T < 30 min.
(D) Progression of resistance sweeps between 0–48 hr shown as distribution of ON cell frequency across populations. Frequency of ON cells (average over 12-hr bins) increases over time for short durations T indicating effectiveness of selective sweeps.
(E) Long-term growth rates (average over 12-hr bins) show significant increase over time for T = 6, 12 min but not for longer durations.
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4. Figure 3
Single-Cell Analysis of Susceptible Cells under Fluctuating Selection
(A–C) Single-cell measurement of susceptible (OFF) cells in the Sel. strain for (A) T = 3 hr, (B) T = 30 min, and (C) T = 6 min. Data are shown from a typical chamber in each experiment. Error bars show SE among multiple independent populations. Antibiotic killing phases are represented by gray shades. Purple dashed lines, growth rate in non-selective LB medium. Image panels (mCherry channel) show cell morphology after 12 hr of fluctuating selection. Populations in these images have not acquired resistant ON cells; thus, all cells shown here are susceptible OFF cells.
(D and E) Autocorrelation of elongation rate (D) and cell-division rate (E) computed for susceptible cells as the Pearson correlation of measurements at times t and t + τ, shown as a function of τ. Periodicities of both elongation rate and cell-division rate vanish for short periods.
See also Figure S2.
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5. Figure 4
Modeling Cellular Growth Physiology and Memory in a Changing Environment
(A) Periodic average of population growth over all populations for each value of T, using experimental data of Ctl. strains (circles) and model fit (dot-dashed lines). Purple horizontal dashed lines: growth rate in non-selective LB medium.
(B) Long-term growth rates measured (circles) and predicted by different models (curves). Magenta circles: time-averaged g from Figure 4A. Blue circles, effective growth rate of OFF cells from Figure 2B. For the model with memory, a memory kernel timescale of tm = 1 min was used, comparable with timescales for tetracycline to cross cell membranes [26] and for binding/unbinding to ribosomes [27, 28] (see Supplemental Information). Inset: dependence of dg/dt (min–2) on g (min–1) in ODE model for killing (red) and recovery (black) phases.
(C) Simulated population dynamics of selective sweeps based on the single-cell physiological model (see Supplemental Information).
See also Figures S3 and S4.
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