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Single cell kinetics of phenotypic switching in the arabinose utilization system of E. coli.

Fritz G, Megerle JA, Westermayer SA, Brick D, Heermann R, Jung K, Rädler JO, Gerland U - PLoS ONE (2014)

Bottom Line: We find that rapid off-switching does not depend on internal arabinose degradation.We construct such a mutant and confirm the graded response experimentally.Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.

View Article: PubMed Central - PubMed

Affiliation: Arnold Sommerfeld Center for Theoretical Physics and CeNS, Ludwig- Maximilians-Universität München, Munich, Germany ; Center for Integrated Protein Science (CiPSM) at the Department of Biology, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.

ABSTRACT
Inducible switching between phenotypes is a common strategy of bacteria to adapt to fluctuating environments. Here, we analyze the switching kinetics of a paradigmatic inducible system, the arabinose utilization system in E. coli. Using time-lapse fluorescence microscopy of microcolonies in a microfluidic chamber, which permits sudden up- and down-shifts in the inducer arabinose, we characterize the single-cell gene expression dynamics of the araBAD operon responsible for arabinose degradation. While there is significant, inducer-dependent cell-to-cell variation in the timing of the on-switching, the off-switching triggered by sudden removal of arabinose is homogeneous and rapid. We find that rapid off-switching does not depend on internal arabinose degradation. Because the system is regulated via the internal arabinose level sensed by AraC, internal arabinose must be rapidly depleted by leakage or export from the cell, or by degradation via a non-canonical pathway. We explored whether the poorly characterized membrane protein AraJ, which is part of the arabinose regulon and has been annotated as a possible arabinose efflux protein, is responsible for rapid depletion. However, we find that AraJ is not essential for rapid switching to the off-state. We develop a mathematical model for the arabinose system, which quantitatively describes both the heterogeneous on-switching and the homogeneous off-switching. The model also predicts that mutations which disrupt the positive feedback of internal arabinose on the production of arabinose uptake proteins change the heterogeneous on-switching behavior into a homogeneous, graded response. We construct such a mutant and confirm the graded response experimentally. Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.

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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).
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pone-0089532-g005: 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).

Mentions: Bacteria were attached to the surface of a microfluidic flow chamber and expression of the ara system was induced by applying a contiuous flow of minimal medium supplemented with defined concentrations of arabinose. In that, our protocol asserts that extracellular conditions are constant and arabinose is not depleted from the medium. After induction with different arabinose concentrations (0.5%, 0.05% and 0.01%) at t = 0 min, we followed gene expression dynamics by fluorescence time-lapse microscopy (Fig. 2A–C) and determined the mean fluorescence (sum over fluorescence values divided by the number of pixels) in individual cells over time (Fig. 2D–F). For each cell, we then estimated the time delay until promoter activation from the fluorescence time series, using the same method as in [23], see also ‘Materials and Methods’. The resulting distributions of delay times in Fig. 2G-I can therefore be directly compared to the analogous distributions for the ΔaraBAD mutant in Fig. 5 of ref. [23].


Single cell kinetics of phenotypic switching in the arabinose utilization system of E. coli.

Fritz G, Megerle JA, Westermayer SA, Brick D, Heermann R, Jung K, Rädler JO, Gerland U - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3935871&req=5

pone-0089532-g005: 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).
Mentions: Bacteria were attached to the surface of a microfluidic flow chamber and expression of the ara system was induced by applying a contiuous flow of minimal medium supplemented with defined concentrations of arabinose. In that, our protocol asserts that extracellular conditions are constant and arabinose is not depleted from the medium. After induction with different arabinose concentrations (0.5%, 0.05% and 0.01%) at t = 0 min, we followed gene expression dynamics by fluorescence time-lapse microscopy (Fig. 2A–C) and determined the mean fluorescence (sum over fluorescence values divided by the number of pixels) in individual cells over time (Fig. 2D–F). For each cell, we then estimated the time delay until promoter activation from the fluorescence time series, using the same method as in [23], see also ‘Materials and Methods’. The resulting distributions of delay times in Fig. 2G-I can therefore be directly compared to the analogous distributions for the ΔaraBAD mutant in Fig. 5 of ref. [23].

Bottom Line: We find that rapid off-switching does not depend on internal arabinose degradation.We construct such a mutant and confirm the graded response experimentally.Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.

View Article: PubMed Central - PubMed

Affiliation: Arnold Sommerfeld Center for Theoretical Physics and CeNS, Ludwig- Maximilians-Universität München, Munich, Germany ; Center for Integrated Protein Science (CiPSM) at the Department of Biology, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany.

ABSTRACT
Inducible switching between phenotypes is a common strategy of bacteria to adapt to fluctuating environments. Here, we analyze the switching kinetics of a paradigmatic inducible system, the arabinose utilization system in E. coli. Using time-lapse fluorescence microscopy of microcolonies in a microfluidic chamber, which permits sudden up- and down-shifts in the inducer arabinose, we characterize the single-cell gene expression dynamics of the araBAD operon responsible for arabinose degradation. While there is significant, inducer-dependent cell-to-cell variation in the timing of the on-switching, the off-switching triggered by sudden removal of arabinose is homogeneous and rapid. We find that rapid off-switching does not depend on internal arabinose degradation. Because the system is regulated via the internal arabinose level sensed by AraC, internal arabinose must be rapidly depleted by leakage or export from the cell, or by degradation via a non-canonical pathway. We explored whether the poorly characterized membrane protein AraJ, which is part of the arabinose regulon and has been annotated as a possible arabinose efflux protein, is responsible for rapid depletion. However, we find that AraJ is not essential for rapid switching to the off-state. We develop a mathematical model for the arabinose system, which quantitatively describes both the heterogeneous on-switching and the homogeneous off-switching. The model also predicts that mutations which disrupt the positive feedback of internal arabinose on the production of arabinose uptake proteins change the heterogeneous on-switching behavior into a homogeneous, graded response. We construct such a mutant and confirm the graded response experimentally. Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.

Show MeSH
Related in: MedlinePlus