Research ArticleMICROBIOLOGY

Stochastic transcriptional pulses orchestrate flagellar biosynthesis in Escherichia coli

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Science Advances  05 Feb 2020:
Vol. 6, no. 6, eaax0947
DOI: 10.1126/sciadv.aax0947
  • Fig. 1 Stochastic pulsing of flagellar promoters.

    (A) The flagellar operons are organized in a transcriptional three-tiered cascade and are accordingly labeled class I (green), class II (red), or class III (blue). For simplicity, our diagram omits various branching and feedback regulatory pathways (such as the anti–sigma factor FlgM). (B) A microfluidic device (“mother machine”) constrains cells to grow in narrow linear tracks (fig. S1). As the cells grow and divide, they are flushed away at the open end of the tracks by a constant flow of medium, which ensures chemostatic growth. We monitor the cell confined at the bottom (the “mother cell,” red) to build a lineage over multiple cell divisions. (C) Kymographs illustrating the pulsating dynamics of flagellar promoters. Each kymograph shows fluorescence (false color, orange) from a strain harboring a YFP transcriptional fusion to a specific flagellar promoter from either class I (top), II (middle), or III (bottom). Cells were grown in Mops-rich defined medium at 34°C, and fluorescence images from each cell were acquired every 10 min. White dots indicate the location of the mother cell in each frame, which was identified via a constitutively expressed mCherry marker. Cells with the promoter encoding the class I master regulator (top) are continuously fluorescent. By contrast, class II (middle) and class III promoters (bottom) stochastically switch between active and inactive transcriptional states over multiple divisions.

  • Fig. 2 Promoters across classes show distinct dynamics but pulse simultaneously within their own class (II or III).

    (A) Typical activity of the sole class I promoter, flhD, which controls the expression of the master regulator (green). Promoter activity is quantified by taking the cell growth–corrected time derivative of the associated fluorescence signal (see the “Estimation of promoter activity from time-lapse data” section). (B) (Top) Activity of fliF (dark red) and fliA (bright red) promoters within the same cell, representative of class II pulsing dynamics. (Bottom) Correlation between two class II gene reporters in the same cell as determined by flow cytometry. Each strain harbors a reference reporter consisting of the fliF promoter and CFP and a second class II promoter fused to YFP. The control promoter is a synthetic constitutive promoter. (C) Activity of fliC (dark blue) and motA (light blue) promoters within the same cell, representative of class III pulsing dynamics. (Bottom) Correlation between two class III gene reporters in the same cell as determined by flow cytometry. Similar to (B), each strain harbors a reference reporter consisting of the fliC promoter and CFP and a second class III promoter fused to YFP. The control promoter is again a synthetic constitutive promoter. (D) Normalized autocorrelation function of flagellar promoter activity of classes I (green), II (red), and III (blue), estimated from the activity of flhD, fliA, and tar promoters, respectively (see the “Correlation analysis” section). For each promoter, the autocorrelation was estimated from 50 lineages, each at least 40 generations long. (E) Cumulative distribution of the pulse on durations, plotted as log(1 − CDF). The distributions for fliF (class II, red) and fliC (class III, blue) are shown. Durations were estimated from 50 lineages, each at least 60 generations long. (F) Cumulative distribution of the pulse off durations, plotted as log(1 − CDF). The distributions for fliF (class II, red) and fliC (class III, blue) are shown. Durations were estimated from 50 lineages, each at least 60 generations long.

  • Fig. 3 Class II pulses do not require regulation of FlhDC transcription or translation by endogenous regulators.

    (A) (Top) Endogenous expression of FlhDC is driven by the class I promoter, which is regulated by multiple transcription factors (TF) (53). In addition, translation of FlhDC is regulated by several sRNAs interacting with the 5′UTR of the FlhDC transcript (top left). We replaced the native class I promoter with a synthetic constitutive promoter (Pro4)—in addition, we altered the 5′UTR so that the synthetic RBS, which drives FlhDC translation, is insensitive to sRNA regulation (top right). (Bottom) Typical class II promoter dynamics (fliF promoter, red) and wild-type (bottom left) and “constitutive” (bottom right) FlhDC promoters. (B) Cumulative distribution function (plotted as 1 − CDF) of class II promoter activity amplitudes from wild-type (solid red circle) and constitutive strains (open red circle). For each strain, we analyzed 50 lineages, each at least 30 generations long. (C) Normalized autocorrelation function of class II promoter activity, wild type (dark red), and synthetic constitutive (light red). For each strain, we analyzed 50 lineages, each at least 30 generations long. (D) Simultaneous measurements of fluorescence signal from the class I reporter (green) and activity of the class II promoter fliFp (red) within the same cell. The fluorescence signal from the class I reporter is a proxy for the concentration of proteins produced from the FlhDC promoter, while the class II promoter activity is the time derivative of the fluorescence signal from the class II reporter, which is a proxy for transcription from that promoter. Typical examples of wild-type (upper) and ΔYdiV cells (lower), along with the Pearson correlation coefficient for the class I and class II signals. For ease of visualization, each signal is normalized so that the minimum value of the signal is 0 and the maximum value of the signal is 1. Pearson correlation coefficient was computed on the raw data before normalization. (E) Normalized histogram showing distribution of correlation coefficients from 100 lineages for wild-type (blue) and ΔYdiV (yellow) strains. Solid lines, kernel density approximations of those distributions (red and purple, respectively). Each lineage is at least 30 generations long. a.u., arbitrary units.

  • Fig. 4 Modulation of class III pulses by posttranslational regulation of the alternative sigma factor, FliA.

    (A and B) Paired measurements of class II and class III within the same cells. We compared fluctuations in class II fluorescence signal (red, fliFp), a proxy for the concentration of proteins produced from class II promoters, to the activity of a class III promoter (fliCp, blue). Typical examples, (A) wild-type cells, and (B) a ΔFlgM mutant that constitutively expresses the master regulator, FlhDC, to bypass any transcriptional feedback. (C) Mean class III activity as a function of the class II reporter concentration in cells harboring fliF and fliC promoter reporters. Binned average of single-cell measurements and wild-type (circles) and ΔFlgM (diamonds) strains; ΔFlgM mutant, same as in (B). For each strain, we analyzed 25 lineages, each at least 50 generations long.

  • Fig. 5 YdiV underlies the dynamics of a pulsatile, bet-hedging circuit.

    (A and B) Input-output relationship between class I and class II. We generated strains where class I expression was driven by a synthetic promoter of different strength (P1 to P7, see the “Construction of strains with constitutively expressed FlhDC” section and table S3 from the Supplementary Materials) and plotted the class II (fliFp) promoter activity in those strains as a function of class I expression. For each strain, we divided class I reporter levels into five logarithmically spaced bins and plotted the mean input (i.e., class I) against the median of the corresponding output (i.e., class II) in each bin. Error bars indicate the 15 and 85% bounds of class II activity for each bin. Contour plots depicting the density of the paired class I and class II data for each strain are also shown. (A) shows ΔYdiV mutant strains, while (C) shows strains with wild-type YdiV. Dashed red line indicates mean class II promoter activity of strain with wild-type FlhDC promoter without [in (A)] and with [in (B)] YdiV. The inset in (A) highlights strains with promoters P3, P4, and P5 that have substantially overlapping class I expression yet show distinct class II outputs. For each strain in (A) and (B), we analyzed at least 50 lineages, each at least 20 generations long. (C and D) Distribution of class II promoter activities for each strain (C) without or (D) with YdiV. Shaded distributions correspond to P1, P3, P5, and P7. (E) Class II promoter activity distributions for P4 strain without YdiV (violet) and P5 strain with YdiV (blue). Both strains have similar mean class II activities (7.1 versus 7.4), but in the presence of YdiV, the class II activity distribution becomes considerably wider. (F) Commitment versus bet-hedging behavior in ΔYdiV and wild-type cells. Each strip is a typical kymograph of a strain harboring a class II promoter reporter expressing FlhDC from synthetic promoters.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/6/eaax0947/DC1

    SUPPLEMENTAL STRAIN CONSTRUCTION METHODS

    Fig. S1. Schematic of the microfluidic device.

    Fig. S2. Flagellar gene expression is heterogeneous when cells are grown in liquid suspension.

    Fig. S3. Flow cytometry analysis of flagellar gene expression for cells grown in liquid culture.

    Fig. S4. The cis-YFP reporter in the FlhDC operon does not affect class II gene expression.

    Fig. S5. Estimation of promoter activity from time-lapse data.

    Fig. S6. Class I (flhDp) promoter activity resembles a constitutive promoter.

    Fig. S7. Promoters within the same class show high correlation.

    Fig. S8. Calibration of class I reporter for mut4 and T7 RBS.

    Fig. S9. An insertion element mutation in the FlhDC regulatory region results in high homogenous expression of flagellar genes.

    Fig. S10. Effect of flagellar gene deletions on class II pulses.

    Fig. S11. Constitutively expressed YdiV can restore class II promoter pulses in a ∆ydiV mutant of E. coli.

    Fig. S12. Comparison of Salmonella and E. coli class II and III gene expression.

    Table 1. List of plasmids.

    Table 2. List of strains.

    Table 3. List of the combination of promoter and RBS used to control class I expression and the notation used in this work to reference them.

    References (6676)

  • Supplementary Materials

    This PDF file includes:

    • SUPPLEMENTAL STRAIN CONSTRUCTION METHODS
    • Fig. S1. Schematic of the microfluidic device.
    • Fig. S2. Flagellar gene expression is heterogeneous when cells are grown in liquid suspension.
    • Fig. S3. Flow cytometry analysis of flagellar gene expression for cells grown in liquid culture.
    • Fig. S4. The cis-YFP reporter in the FlhDC operon does not affect class II gene expression.
    • Fig. S5. Estimation of promoter activity from time-lapse data.
    • Fig. S6. Class I (flhDp) promoter activity resembles a constitutive promoter.
    • Fig. S7. Promoters within the same class show high correlation.
    • Fig. S8. Calibration of class I reporter for mut4 and T7 RBS.
    • Fig. S9. An insertion element mutation in the FlhDC regulatory region results in high homogenous expression of flagellar genes.
    • Fig. S10. Effect of flagellar gene deletions on class II pulses.
    • Fig. S11. Constitutively expressed YdiV can restore class II promoter pulses in a ∆ydiV mutant of E. coli.
    • Fig. S12. Comparison of Salmonella and E. coli class II and III gene expression.
    • Table 1. List of plasmids.
    • Table 2. List of strains.
    • Table 3. List of the combination of promoter and RBS used to control class I expression and the notation used in this work to reference them.
    • References (6676)

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