Kinetics of large-scale chromosomal movement during asymmetric cell division in <i>Escherichia coli</i>

<div><p>Coordination between cell division and chromosome replication is essential for a cell to produce viable progeny. In the commonly accepted view, <i>Escherichia coli</i> realize this coordination via the accurate positioning of its cell division apparatus relative to the nucleoids. However, <i>E</i>. <i>coli</i> lacking proper positioning of its cell division planes can still successfully propagate. Here, we characterize how these cells partition their chromosomes into daughters during such asymmetric divisions. Using quantitative time-lapse imaging, we show that DNA translocase, FtsK, can pump as much as 80% (3.7 Mb) of the chromosome between daughters at an average rate of 1700±800 bp/s. Pauses in DNA translocation are rare, and in no occasions did we observe reversals at experimental time scales of a few minutes. The majority of DNA movement occurs at the latest stages of cell division when the cell division protein ZipA has already dissociated from the septum, and the septum has closed to a narrow channel with a diameter much smaller than the resolution limit of the microscope (~250 nm). Our data suggest that the narrow constriction is necessary for effective translocation of DNA by FtsK.</p></div

Chromosomal movement reported by DAPI and HupA-mCherry labels.

<p>A: Time-lapse images showing DAPI (top row) and HupA-mCherry labelled chromosomes (middle row). Arrows point to location of cell division plane. Phase images for the same cell are shown in the bottom row. Scale bar is 2 μm. B: Normalized intensity from DAPI and HupA-mCherry labels during cell division for the cell shown in panel A. Dashed horizontal line corresponds to the expected value for both intensities at the end of the division. C: Initial normalized intensity vs final normalized intensity from DAPI label (N = 46). Dashed lines correspond to the expected values for the final normalized intensity. In this plot data from cells without Z-ring marker (strain JM30) and with induced ZipA-GFP Z-ring marker (strain MB16) have been combined. The data from two strains are plotted separately in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006638#pgen.1006638.s004" target="_blank">S3 Fig</a> in Supplemental Materials. These data show no significant differences between the two strains.</p

Chromosomal movement reported by SYTOX-Green and HupA-mCherry labels.

<p>A: Time-lapse images showing SYTOX-Green (top row) and HupA-mCherry labelled chromosomes (middle row) in a representative cell (strain JM30). Arrows point to location of cell division plane. Phase images for the same cell are shown in the bottom row. Scale bar is 2 μm. B: Normalized intensity from SYTOX-Green and HupA-mCherry labels during cell division for the cell shown in panel A. Dashed horizontal line corresponds to the expected value for both intensities at the end of the division. C: Initial normalized intensity vs final normalized intensity from SYTOX-Green label. Dashed lines correspond to the expected values for the final normalized intensity and solid diagonal to no change in intensity. N = 19.</p

Correlations between translocation timing, width of the constriction, and translocation speed.

<p>A: Normalized width of the Z-ring when translocation initiates. For normalization, the width of unconstricted Z-ring is used. Mean and std of the distribution are shown. Strain MB16. N = 24. B: Translocation start time vs. translocation speed. Solid line is a linear fit (y = 32 x+2000; R = 0.37).</p

Quantification of DNA movement during translocation.

<p>A: Estimated amount of DNA in the smaller daughter compartment in the beginning and end of translocation. The amount of DNA is given in genome units (4.6 Mb). Arrows pointing to the right indicate divisions in which the DNA amount in the smaller daughter cell increases and arrows pointing to the left are those where the amount decreases. No change in DNA amount is marked by solid diagonal line. B: Distribution of the DNA amount that has crossed the division plane during translocation. DNA amounts are given in genome units. Positive amounts correspond to DNA moving into the smaller daughter compartment and negative amounts moving out from it. C: Distribution of translocation speeds. N = 46 for all the panels. Data from strains JM30 and MB16 is combined.</p

No large-scale chromosomal translocation in cells carrying additional FtsK K997A mutation.

<p>A: Time-lapse images showing DAPI (top row) and HupA-mCherry labelled chromosomes (middle row). Arrows point to location of cell division plane. Phase images for the same cell are shown in the bottom row. Scale bar is 2 μm. B: Normalized intensity from DAPI and HupA-mCherry labels during cell division for the cell shown in panel A. C: Initial vs final normalized intensity from DAPI label in strain JM38 (Δ<i>slmA</i> Δ<i>minC</i> Δ<i>zapB ftsK K997A</i>). Only cells whose length is smaller than 10 μm have been analyzed. Solid diagonal line corresponds to cases where the DNA amount in smaller daughter compartment remains unchanged during division. Dashed horizontal lines correspond to final normalized intensities of 0, 25, 33 and 50%. N = 13.</p

Chromosomal movement across the septum in asymmetrically dividing Δ<i>slmA</i> Δ<i>minC</i> Δ<i>zapB</i> cells.

<p>A: Time-lapse images showing HupA-mCherry labelled chromosomes (top row) and overlay of HupA-mCherry and phase images in an asymmetrically dividing cell (middle row). Bottom row shows intensity line profiles along the long axes of this cell. The black line is for phase and red for the HupA-mCherry profile. Time zero corresponds to the appearance of constrictions in phase images. Arrows in top row of images point to location of cell division plane. Strain JM30. Scale bar is 2 μm. B: Schematics showing calculation of normalized intensity. <i>I</i>_<i>1</i> and <i>I</i>_<i>2</i> are the integrated fluorescent intensities of the nucleoid label on the smaller and larger daughter side of the cell division plane, respectively. C: Normalized intensity from HupA-mCherry label during cell division for the cell shown in panel A. D: Initial normalized intensity at the time when constriction appears vs. final normalized intensity at the time when cells divide for 25 cells. Horizontal dotted lines show expected final normalized intensity values. Solid diagonal line marks no change in normalized intensity during division.</p

Translocation timing.

<p>A: Distribution of times when translocation starts as inferred from DAPI label. The times in all panels are referenced relative to the time when the ZipA-GFP label dissociates from the septum. Mean and std of distribution are indicated. Strain MB16. N = 24 for all panels. B: Distribution of times when translocation ends as inferred from DAPI label. C: Duration of translocation for individual cells as inferred from DAPI label. D: Distribution of times when HupA-mCherry normalized intensity stops to change. Note that HupA-mCherry normalized intensity starts to change at the same time as DAPI normalized intensity. E: Timing of translocation for individual cells as inferred from HupA-mCherry label.</p

Determination of translocation timing based on Z-ring and phase contrast images.

<p>A: Kymographs of DAPI, HupA-mCherry, ZipA-GFP labels and phase contrast images along the long axes of a representative cell. Red corresponds to high and blue to low intensity. Black areas are outside the cell. Time zero corresponds to the first frame when no ZipA-GFP accumulation is present at the constriction. Dashed lines in the bottom of every kymograph show the time interval corresponding to traces in panels B-D. Strain MB16. B: Normalized intensity from DAPI and HupA-mCherry labels as a function of time for the same cell. C: Width of the Z-ring as a function of time. The width is measured along the short axes of the cell. D: Modulation of phase contrast images at the constriction as a function of time. The inset shows determination of the modulation from phase contrast profiles along the long axes of the cell.</p

Model relating translocation efficiency and timing.

<p>Left: Although FtsK is recruited to Z-ring early, DNA translocation starts when the septum is closing because only then does DNA from the nucleoid become accessible to FtsK hexamers. Moreover, for almost open septum the processivity is low because the FtsK pump(s) has a low probability of rebinding to DNA once the two dissociate. The direction of pumping is to the compartment containing <i>oriC</i>. Right: In late stages of division constriction forms a small channel which acts as a diffusion barrier to proteins. The channel does not close before DNA is cleared from it. The processivity of DNA transport in this channel is high because FtsK and DNA remain in close proximity. When the two dissociate from each other then they quickly rebind. Also, FtsK may not be able to reverse its direction when confined by the channel.</p