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. 2017 Mar 31;6:e21763. doi: 10.7554/eLife.21763

Figure 1. Most replisome subunits exchange frequently with the diffusing pool.

(A) Model illustrating the architecture of a replisome at the E. coli replication fork. (B) Representative fluorescence images of FRAP experiments for the Pol III α subunit and the DnaB helicase. Cell boundaries shown as white lines, red circle shows the location of the bleached focus. (C) Representative examples of the FRAP curves for Pol III α subunit (N = 48) and DnaB (N = 96). Red line shows a reaction-diffusion model fit to the data, dashed grey lines show SE for the model. Dashed blue line represents the estimated maximum possible fluorescence recovery after correcting for photobleaching. (D) Analysis summary of the replisome by FRAP. Bars represent average bound-times. Red squares represent level of recovery normalised to the intensity before bleaching. Dashed blue line represents maximum possible fluorescence recovery. It was not possible to estimate the bound-time for DnaB. Error bars represent SE.

DOI: http://dx.doi.org/10.7554/eLife.21763.003

Figure 1.

Figure 1—figure supplement 1. Artificial elongation of cells by cephalexin treatment does not interfere with DNA replication or protein expression.

Figure 1—figure supplement 1.

(A) Representative images of non-treated (M9-Gly) or cells treated with 40 µg/ml of cephalexin (M9 Gly + Cephalexin) are shown. The position of the ori1 locus (mapped at 3908 kb in the chromosome), labelled by TetR-mCerulean bound to a tetO array, is shown in red, while the position of the replisome component SSB labelled with YPet is shown in green. Top panels do not show the phase contrast. Scale bar represents 2 µm. A table summarizes analysis of this data, showing the number of ori1 and SSB foci per cell, and the ratio between them. (B) Distribution of the total intensity of DAPI signal against cell length in cells untreated (blue dots) and treated with cephalexin for one hour (red dots). Ethanol fixation was used to ensure homogenous permeability to the dye. Fitting to a linear model is shown in the respective colours. The inset shows the distribution of mean intensities per pixel for both conditions. (C) Distribution of the total intensity of ε-YPet signal against cell length in cells untreated (blue dots) and treated with cephalexin for one hour (red dots). Fitting to a linear model is shown in the respective colours. The inset shows the distribution of mean intensities per pixel for both conditions.
DOI: http://dx.doi.org/10.7554/eLife.21763.004

Figure 1—figure supplement 2. Minimal contribution of YPet photoblinking during FRAP.

Figure 1—figure supplement 2.

(A) Representative fluorescence images of a cell carrying a tetO operator array and expressing TetR-YPet. Cells were fixed with formaldehyde to avoid protein exchange. Cell boundaries are represented with a white line. The point of localized bleaching is shown with a red circle. (B) Average distribution of fluorescence recovery after photobleaching of 49 cells. Note that intensity increase after bleaching is minimal (<5% of the total intensity), consistent with stochastic fluctuations and experimental measurement error. Error bars represent SE.
DOI: http://dx.doi.org/10.7554/eLife.21763.005

Figure 1—figure supplement 3. Growth rate and replication time of E. coli in our experimental conditions.

Figure 1—figure supplement 3.

(A) Growth curve of AB1157 in M9-Glycerol at 22°C is shown. Samples were taken every hour for 7 hr. (B) Distribution of the number of spots per cell of a strain carrying ε-YPet grown in M9-Glycerol at 22°C (N = 1403 cells). We estimated the replication time by taking into account the generation time and the number of cells with spots. We made the assumption that initiation of DNA replication occurs at cell birth to account for the uneven distribution of cell ages in the population. (C) Representative images obtained from a time-lapse experiment of a strain carrying SSB-YPet grown on a 1% agarose pad in M9-Glycerol at 22°C. Pictures were taken at 10 min intervals. Replication time was determined from the time point of first appearance of SSB-YPet spot to its subsequent disappearance (N = 56 cells). The average of the two methods (150 min) is reported in the main text.
DOI: http://dx.doi.org/10.7554/eLife.21763.006