Dynamics of the Bacterial Chromosome - Structure and Function

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Time is indicated in hours. Orange outlines the cell contour. Bottom: cartoon illustrations; black strands indicate newly replicated DNA. Indicated are the ridge of the bundle green dashed line , the oriC and dif genomic loci near the origin and terminus of replication red and blue dots respectively , and the bundle width blue line.

Whole-chromosome imaging 11 , 17 , 18 would be an ideal tool to resolve and characterize the fine structure of chromosomes. We set out to overcome these limitations in two ways: First, we used the MreB-protein-inhibitor drug A22 to disrupt the formation of new cytoskeleton for the cell, which led to cells that grew wider in size, while staying alive in a fully physiologically active state 22 Supplementary Fig.

Interestingly, upon a two-fold widening of the cell, the single E. This topology was consistently observed through different imaging techniques such as wide-field epifluorescence and 2D and 3D Structured Illumination Microscopy SIM Fig. These images of an open ring-like geometry confirmed that two chromosome arms flanking the origin of replication in E. Note that the toroidal geometry is not trivial, since a priori, cell widening could have been expected to lead to a homogeneously spread-out globular cloud of DNA 26 , an unaltered ellipsoid 2 , or a stiff arc By contrast, upon treatment for a short time with drugs such as rifampicin which blocks transcription by inhibiting RNA Polymerase or ciprofloxacin which impedes the homeostasis of supercoiling through inhibiting TopoIV and gyrase activity , or upon induction of the stationary phase, the chromosomes collapsed and generally lost the torus topology Supplementary Fig.

All of this indicates that it is a general feature for a single chromosome in widened E. We conclude that the torus topology is maintained by active physiological processes, and hence serves as an excellent model object for resolving the organizational principles of a E. The direct visualization of the genome allowed us to quantitatively measure the width and length of the E.

Organization and function of the genome

Facilitated by deconvolution which reduced the out-of-focus background intensity in wide-field imaging Fig. The average chromosome contour length was found to be 4. These data provide useful input for future modeling of the polymer structure of the chromosome under weak confinement and volume exclusion in the segregation of newly replicated DNA 2 , 11 , The donut-shape chromosomes were observed to be strikingly non-uniform.

The DNA density was heterogeneous along the circumference, partitioned into blob-like domain structures Fig. Using a custom-made cluster-analysis script , Supplementary Figs.

Next, we quantified the DNA length L measured in base pairs contained in each cluster based on fluorescence intensity, yielding values from kbp to above 3 Mbp Fig. Domain distribution within the circular chromosome of E. Top panel, fluorescent image of a toroidal chromosome deconvolved image. Bottom panel, same image with ridge line red , equal-intensity lines thin cyan lines , and blob boundaries yellow. Black line shows the probability density function PDF of a fitted lognormal distribution. Circles indicate mean calculated for all cells within a kbp bin size.

Inset shows the same data plotted on linear scales. In order to quantify the average DNA density as a function of the genomic sequence coordinate, we mapped the HUmYpet fluorescence intensity along the ridges of the donut-shape chromosomes Supplementary Figs. Next, we mapped the contour coordinates onto the genome sequence by constructing a cell-average cumulative density function that starts and ends at the L3 foci, which allowed physical positioning of the genomic loci including oriC and dif sites where DNA replication initiates and terminates, respectively onto the torus Fig.

DNA density mapping along the circular E. The arrow indicates the direction of density mapping. Each line indicates a single chromosome. An example chromosome density is highlighted in black.

Bacterial Chromosome Organization and Segregation

Blue arrows indicate local maxima peaks and local minima valleys in this example curve. Marks indicate the measured positions of the R3 locus and global minimum I min , and the predicted positions of oriC and dif sites. Error bars indicate s. Dark and light shading indicates s. Inset: schematic illustrating the DNA density distribution along a typical circular E. Note that in this strain left and right arms can be distinguished less well due to symmetry Fig.

Local maxima and minima are identified as in c. This yielded the average DNA density profile, which displays a pronounced M-shape curve with a very deep minimum located at the dif locus and a second, less deep yet well developed, minimum at the oriC locus Fig. Interestingly, at the global density minimum which consistently resides near the dif site Fig. These oriC and dif loci are connected by the DNA-dense left and right arms, which show a slight asymmetry with a somewhat higher DNA density peak in the left arm.

The average M-shape was also conserved in experiments with multiple different fluorescent labels Supplementary Fig. Individual DNA density plots of single chromosomes cf. As shown in Fig. By contrast, less pronounced domain centres and boundaries were found to distribute more evenly throughout the genome Fig. This led us to hypothesize that different mechanisms may be at play in defining the chromosomal domain structure in E. Whereas broadened cells without MatP also exhibited toroidal chromosomes Fig.

Interestingly, local density peaks and gaps were still observed in individual cells, but they were evenly distributed across the chromosomes with no prominent features in either the oriC or dif sites Fig. Notably, the thin terminal string persisted at different stages of the replication cycle Fig. MatP thus is found to be crucial for the formation of the prominent domain boundaries at both the dif and oriC regions, likely for promoting accessibility of these sites to proteins involved in the spatiotemporal regulations of DNA segregation and cell division 31 , Although the average DNA density distribution clearly showed one peak at each of the two chromosomal arms Fig.

We examined the origin of the secondary domain boundaries by quantifying their distribution in the presence and absence of the nucleoid-associated proteins NAPs HU, H-NS, and Fis, which function as global transcription regulators Given that HU and Fis are transcription activators that localize sequence-nonspecifically throughout the genome Supplementary Figs. These data, and our observations from antibiotic treatments Supplementary Fig.

The strong cell-to-cell variations suggest that these domains are dynamic in nature. Indeed, very pronounced dynamics are apparent in time-lapse imaging of the donut-shape chromosomes.

Self-organised segregation of bacterial chromosomal origins

Our approach allowed us to construct coarse proximity maps of a single genome within a single live cell over time Fig. The prominent domain boundary at the dif region was very persistent, and the weaker one at oriC region was present as well in most of the frames. These rough proximity maps are somewhat reminiscent of Hi-C maps that describe the contact frequencies between genomic loci in an ensemble of chemically fixed cells 8 , 9 , 18 , but are also characteristically different as they measures the real-time toroidal distance within a single genome in a live cell.

Temporal dynamics of the circular chromosome of E. Time stamps are in minutes. The last frame is the time-average. Color bar indicates the level of spatial proximity for genomic loci along the circular chromosome. Arrowheads indicate apparent domain boundaries. Top panels, two consecutive frames, the time-average, and standard deviation values. Bottom panel, all 11 time frames. These cross-correlation functions are measured with regard to the DNA density distribution along the contour in, respectively, spatial coordinates c genomic coordinates d , and their morphology e.


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Shown are mean and standard deviation values. The autocorrelation function of the density distributions along both the spatial Fig. Similar time constants for local chromosomal rearrangements were reported in rod-shaped cells 17 , suggesting that the chromosome dynamics that we observe in these circularly shaped cell are depicting genuine chromosome rearrangements. We thus find that whereas the global configuration of the E.

The direct imaging of widened live E. By decoupling DNA replication and cell growth in widened bacteria, we were able to visualize the circular shape of the chromosome in live E. We found that the chromosome was organized in a torus topology with, on average, a lower density at the origin of replication and an ultrathin flexible string of DNA at the terminus of replication.

The organization of the chromosome into a torus geometry is very different from the previously observed stiff arc organization in spheroplasts It is furthermore important to emphasize that our cell-widening method is a gentle method that is not dependent on the use of a temperature or an osmotic shock as previously used Using multiple controls bacterial re-growth after treatment with A22 Supplementary Fig.

The Dynamic Bacterial Chromosome

The thin filamentous structure of the terminus region that we observe was proposed in earlier studies, based on the positioning of local foci relative to the cell shape in slowly growing AB cells 2 , Contradicting results that were obtained in the MG strain, however, claimed instead that the terminus was compacted 13 and suggested that an extended filament might have been specific to the AB strain used in the earlier experiments. Based on our direct imaging, however, we clearly observe, that the terminus is significantly extended into a filament in both strains Figs.

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Our observation suggests that the discrepancy between the previous observations may have been caused by differences in the cell cycle. Previous studies typically used replicating cells, which can lead to cells being in various growth phases with a varying number of chromosomes. For actively replicating cells, the flexible Ter region can be localized to the cell centre due to its interaction with the divisome components 32 , which can lead to an appearance of a self-interacting domain.

To resolve the finer details of chromosome organization, it is therefore important to have the cells synchronized and the chromosome number fixed—as our single-cell studies also show. At the single-cell level, we found the chromosome to be strikingly heterogeneous and dynamic. Furthermore it is interesting to note that the number of six domains was proposed by the macrodomain model 4. Unlike previous studies on HDRs that only reported snapshot images, thus leaving the dynamic behavior unknown, our time-lapse study showed that domains undergo major dynamic rearrangements, splitting and merging and changing position at a minute timescales.

In contrast to the macrodomain model, we found the domain borders to be highly dynamic. The blob number was also influenced by the deletion of MatP proteins in cells, indicating that they regulate long-range interactions. Persistent domain boundaries at the origin and terminus of replication were found to be induced by MatP protein. In a recent Hi-C study 8 , Lioy et al. While our data clearly show that MatP is of central importance in organizing the chromosomal structure near Ter and Ori, the detailed nature of these interactions remains unclear and calls for further research.

We also tested the role of major NAPs in structuring the chromosome. While the deletion of H-NS had, surprisingly, no influence, which may possibly be due to the upregulation of StpA protein 36 , we found that transcription regulators HU and Fis induce weak transient domain boundaries throughout the genome. Recent in vitro studies showed that HU and Fis proteins increase the dynamics as well as decrease the stiffness upon binding to supercoiled DNA Such additional mechanisms may also be at play when forming transient boundaries by NAPs. English Choose a language for shopping.

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