Dynamical determinants of drug-inducible gene expression in a single bacterium

Abstract

A primitive example of adaptation in gene expression is the balance between the rate of synthesis and degradation of cellular RNA, which allows rapid responses to environmental signals. Here, we investigate how multidrug efflux pump systems mediate the dynamics of a simple drug-inducible system in response to a steady level of inducer. Using fluorescence correlation spectroscopy, we measured in real time within a single bacterium the transcription activity at the RNA level of the acrAB-TolC multidrug efflux pump system. When cells are exposed to constant level of anhydrotetracycline inducer and are adsorbed onto a poly-L-lysine-coated surface, we found that the acrAB-TolC promoter is steadily active. We also monitored the activity of the tet promoter to characterize the effect of this efflux system on the dynamics of drug-inducible transcription. We found that the transcriptional response of the tet promoter to a steady level of aTc rises and then falls back to its preinduction level. The rate of RNA degradation was constant throughout the transcriptional pulse, indicating that the modulation of intracellular inducer concentration alone can produce this pulsating response. Single-cell experiments together with numerical simulations suggest that such pulsating response in drug-inducible genetic systems is a property emerging from the dependence of drug-inducible transcription on multidrug efflux systems.

FIGURE 1

Real-time acrAB promoter activity (RNA concentration) in four wild-type cells (Frag1B) induced with 400 ng/ml aTc and one cell without aTc (flat trace). Cells were immobilized on polylysine-coated surfaces. Error bars represent uncertainties in the fit parameters extracted from the autocorrelation function (Methods).

FIGURE 2

Real-time tet promoter activity from 10 single cells induced at (A), 200 ng/ml aTc, (B), 400 ng/ml aTc, and (C), 1 μg/ml aTc. Error bars represent uncertainties in the fit parameters extracted from the autocorrelation function. (D) Average profiles of ms2-RNA concentration from 10 single cells. No induction (light gray), 200 ng/ml aTc (gray), 400 ng/ml aTc (dark gray), and 1 μg/ml aTc (black). Cells were immobilized on polylysine-coated surfaces. Error bars represent the standard deviations from the ms2-RNA concentration distributions across a population of 10 cells at a given time point.

FIGURE 3

RNA decay in individual E. coli cells. DH5αPRO cells induced with 1 μg/ml aTc. Transcription was blocked with the addition of rifampicin at various time points after induction. Decay of RNA concentration in a single cell exposed to 500 μg/ml of rifampicin at 30 min after induction (•). Exponential fit with a decay constant of 0.22 min⁻¹ (solid line). Error bars represent uncertainties in the fit parameters of the associated autocorrelation functions (Methods). (Inset) RNA degradation rate as a function of time from eight cells (dashed line; average degradation rate, 0.26 min⁻¹). Error bars represent fitting uncertainties in the decay constant.

FIGURE 4

Model of inducible transcription in a living cell immobilized on a polylysine-coated surface. (A) Simple reaction scheme of efflux-mediated inducible transcription. T, R, and O represent aTc molecules, TetR dimers, and operator sites, respectively. The tet promoter has two identical operators. RT, ORT, RT₂, and ORT₂ stand for TetR:aTc, O:TetR:aTc, TetR:aTc₂, and O:TetR:aTc₂ complexes, respectively. K₀, K₁, K₂, and K_T are binding constants (Supplementary Material). (B) Simulated RNA profiles (solid lines) in wild-type cells for various aTc induction levels: 200 ng/ml (light shaded), 400 ng/ml (shaded), and 1000 ng/ml (dark shaded). Experimental data from Fig. 2 D (•). Simulated RNA profile (dashed line) for steady intracellular [aTc] = 400 ng/ml (μ = 0 s⁻¹). Experimental data from Fig. 5 A (▪).

FIGURE 5

Efflux-mediated inducible transcription. (A) Concentration profiles of ms2-RNA transcripts from an inducible tet promoter in a Frag1A mutant cell (ΔacrAB) without aTc (shaded) and with 400 ng/ml aTc (solid). Wild-type Frag 1B cell with 400 ng/ml (dark shaded). (B) Tet promoter activity with sequential inductions in four nondividing wild-type cells (Frag1B). Cells were first induced with 100 ng/ml aTc at time zero followed by a second induction with 1 μg/ml aTc at time 60 min after first induction. Arrows indicate times of induction. Error bars represent uncertainties in the fit parameters extracted from the autocorrelation function.