Escherichia coli Chromosomal Loci Segregate from Midcell with Universal Dynamics

Abstract

The structure of the Escherichia coli chromosome is inherently dynamic over the duration of the cell cycle. Genetic loci undergo both stochastic motion around their initial positions and directed motion to opposite poles of the rod-shaped cell during segregation. We developed a quantitative method to characterize cell-cycle dynamics of the E. coli chromosome to probe the chromosomal steady-state mobility and segregation process. By tracking fluorescently labeled chromosomal loci in thousands of cells throughout the entire cell cycle, our method allows for the statistical analysis of locus position and motion, the step-size distribution for movement during segregation, and the locus drift velocity. The robust statistics of our detailed analysis of the wild-type E. coli nucleoid allow us to observe loci moving toward midcell before segregation occurs, consistent with a replication factory model. Then, as segregation initiates, we perform a detailed characterization of the average segregation velocity of loci. Contrary to origin-centric models of segregation, which predict distinct dynamics for oriC-proximal versus oriC-distal loci, we find that the dynamics of loci were universal and independent of genetic position.

Figure 1

Cellular-scale models for chromosome structure. Schematic models of the nucleoid with a left-right filament structure (A) and an ori-ter filament structure (B). The left (right) arm of the chromosome is green (orange). oriC is shown in red and ter is in purple. From top to bottom, the cells in each stack represent chromosome structure at cell birth, during chromosome replication, and before division. To see this figure in color, go online.

Figure 2

Trajectory alignment. (A) Chromosome map of all labeled genetic loci and the sample cell to visualize normalization of positions to cell-length units, L. $X=0$ indicates midcell, and $X>0$$\left(X<0\right)$ represents the old (new)-pole sides of the cell. (B) An example kymograph. To produce kymographs, cell images were projected along the long axis of the cell and aligned sequentially at midcell. Gray regions indicate cell boundaries determined by the brightfield image. Red pixels indicate a fluorescent signal. (C) Three sample fluorescent tracks from oriC kymographs in physical coordinates display a high level of cell-to-cell variation in localization dynamics. (D) Tracks from (C) are normalized to cell length at each point in time, oriented by cell pole, synchronized by splitting times, and overlaid. This method, when repeated with thousands of tracks, can then be used to generate a 3D histogram of locus positioning throughout the cell cycle. To see this figure in color, go online.

Figure 3

Trajectory histograms. Histograms of synchronized trajectories (see Fig. 1D) throughout the cell cycle for several chromosomal loci. Tracks are oriented with $X>0$ indicating the old-pole side of the cell and $X<0$ indicating the new-pole side. The locus label and number of cells contributing to each histogram are as follows: oriC, N = 3528; L2, N = 2254; R3, N = 1416; ter1, $N=406$. Histograms for all seven loci are included in Fig. S2.

Figure 4

Mean locus position trajectories and histogram of split positions. (A and B) Mean locus trajectories aligned by split time oriented using polar (A) and genomic orientation (B). The observation that all loci lie inside oriC demonstrates the predominance of the oriC-ter nucleoid orientation. Loci are also observed to move toward midcell shortly before the initiation of segregation. The tight spacing of the mean trajectory curves demonstrates that the nucleoid configuration is much more compact than observed in AB1157. The noisiness of the mean trajectory at long and short times is due to the small number of cells with cell cycles significantly exceeding 1 h. (Error regions show the error in the mean assuming all observations are uncorrelated. Note that the mean is less meaningful for ter-proximal loci, as their positioning before and after the split is less precise, as can be seen from the locus occupancy of ter1 shown in Fig. 3.) (C) Histogram of splitting locations for all loci in all cells when trajectories are aligned by pole orientation (as in A). Regardless of genetic location or locus long-axis positioning at cell birth, all seven loci split near midcell with high fidelity. To see this figure in color, go online.

Figure 5

Whole-nucleoid imaging. (A) Fis-GFP-labeled nucleoids in single cells. Arrows indicate the new pole (or future new poles) of cells. (B) Consensus localization image of labeled nucleoids from 230 complete cell cycles shows a compact nucleoid asymmetrically distributed toward the new cell pole. (C) Mean DNA density in the first frame of the cell cycle oriented by cell pole (where the right side is negative).

Figure 6

Step-size distributions. (A) The step-size distribution is the probability of observing a step size with a lag time of 1 min. The width of the distribution characterizes the stochasticity of the motion, whereas the mean step size characterizes the directedness of the motion. The initiation of segregation results in a weak bias of the stochastic motion in the direction of average motion. The motion of oriC transitions between unbiased motion (red) and weakly biased motion (green), as seen by the shift in the distribution. The oriC (green) and L2 (blue) loci show essentially identical step-size distributions during the first 10 min of motion. (Error regions show the expected counting error.) (B) Time-dependent histogram of oriC step sizes. Aside from an increased spread in step sizes at the time of locus splitting (t = 0), the distribution in step size is quite homogeneous for the times before and after the split, with a zero bias and consistent spread before the split and a similar spread but with small positive bias after the split.

Figure 7

Mean drift velocities. Profiles of the relative drift velocities of sister loci are provided for ori, L2, and ter. At short time periods after the initial splitting of sister loci, all loci appear to have similar drift velocities, consistent with a model for segregation that treats all loci identically. At later time periods, sister oriC loci have larger relative drift velocities, allowing them a larger net separation along the length of the cell. (Error regions show the error in the mean.) To see this figure in color, go online.

Figure 8

Segregation phenotype of the seqA mutant. To perturb DNA structure shortly after replication, we constructed a seqA deletion. (A) $\Delta$seqA has a nearly universal initial drift velocity. (B) The smaller separation for seqA loci implies that all loci segregate with a lower drift velocity. This is observed as the integrated effect of a velocity that is only slightly smaller acting throughout the segregation process.