Figure 1.
Scheme of the arabinose utilization system in E. coli.
Arabinose is imported via the arabinose transporters AraE and AraFGH. If the intracellular arabinose level is sufficiently large, arabinose binds the transcriptional regulator AraC. This complex activates the promoters PE, PFGH, PBAD and PJ, driving expression of araE, araFGH, araBAD and araJ, respectively. The latter two operons encode proteins for arabinose catabolism (AraBAD) and a putative arabinose efflux pump (AraJ). The negative autoregulation of AraC resulting in homeostatic control of its own level is not shown explicitly. Arrows indicate arabinose transport and positive regulation, whereas the T-shaped arrow indicates arabinose metabolization.
Figure 2.
Single-cell switching dynamics from the off- to the on state in the wildtype ara system.
Bacteria of strain MG1655 were induced with 0.5%, 0.05% or 0.01% arabinose at t = 0 min. The image panels show time series of fluorescence images (A, B, C, scale bar: 5 µm). The single cell fluorescence trajectories (D, E, F) show that fluorescence remains negligible until the so-called delay time at which a strong increase starts. Experimental data points are highlighted by red circles and the corresponding fits are shown as red lines. With decreasing arabinose concentration the delay time increases and varies more significantly between bacteria in a population. This effect is shown quantitatively by the distributions of delay times over the populations (G, H, I), which were extracted by fitting a mathematical function describing the gene expression process. Note that at 0.01% arabinose a significant percentage of the bacteria did not switch on the ara system within the experimental observation window. Number of evaluated cells: 63 (0.5% ara), 74 (0.05% ara), 113 (0.01% ara).
Figure 5.
Single-cell induction kinetics of cells with native and constitutive transporter production.
Cells of strain JW1889-3 (A–C, native araE regulation) and JW1889-5 (D–F, constitutive araE expression) containing the reporter plasmid pBAD24-GFP were induced with indicated concentrations of arabinose. Black lines represent the kinetics of the mean fluorescence of individual cells and red circles highlight representative trajectories. Note that some trajectories disappear before the end of the experiment due to detachment of daughter cells after cell division. Red lines are representative fits of our full model of arabinose uptake and gene regulation in Eqs. (1)–(7) to the highlighted experimental trajectories. Number of evaluated cells: 71, 63 and 54 (for 0.2%, 0.05% and 0.01% ara induction of JW1889-3, respectively); 45, 28 and 57 (for 0.2%, 0.002% and 0.001% ara induction of JW1889-5, respectively).
Table 1.
Median delay times for PBAD activation. Values for strain LMG194 were taken from ref. [23].
Figure 3.
Single-cell response dynamics of the ara system upon arabinose down-shift.
Initially, cells of strain MG1655 (A, native ara regulation), BW25113 (B, ΔaraBAD) and JW0386-1 (C, ΔaraBAD and ΔaraJ) containing the reporter plasmid pBAD24-GFP (gfpmut3 under the control of the PBAD promoter) were prepared in the on-state by induction with 0.5% arabinose for 40 min. At this time arabinose was removed by flushing the microfluidic channel with fresh medium without arabinose. (upper panels) Black lines are experimental fluorescence trajectories, the red circles highlight the data for representative cells, and the bold red lines correspond to the corresponding fits by the model [Eqs. (1)–(7)] under pulsed addition of arabinose. FU: fluorescence units. (lower panels) Red lines show the corresponding dynamics of PBAD promoter activities in individual cells as inferred from the model. Number of evaluated cells: 62 (strain MG1655), 55 (strain BW25113), 51 (strain JW0386-1). For corresponding dynamics without arabinose removal see Fig. S3.
Figure 4.
Theoretical gene expression kinetics for native and constitutive transporter production.
(A) Fluorescent GFP dynamics and (B) PBAD promoter activity for native transporter regulation. (C) Fluorescent GFP dynamics and (D) PBAD promoter activity for constitutive transporter production. Different external arabinose concentrations are represented by various arabinose uptake velocities Vupt. To illustrate stochastic variability in the initial number of uptake proteins, trajectories are shown for 300, 260, 220 and 180 AraE molecules at each Vupt. In all cases, the corresponding number of initial AraE molecules of the shown curves decreases from left to right.
Table 2.
Mean and standard deviation of delay time τd and protein expression rate αp distribution.