ABSTRACT The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in

widened _E. coli_ cells. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin

to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for

replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of

MID-CELL MIGRATION OF THE CHROMOSOMAL TERMINUS IS COUPLED TO ORIGIN SEGREGATION IN _ESCHERICHIA COLI_ Article Open access 18 November 2023 CHROMOSOME ORGANIZATION SHAPES REPLISOME DYNAMICS

IN _CAULOBACTER CRESCENTUS_ Article Open access 24 April 2024 INTRODUCTION DNA replication is vital for the reproduction of all organisms and accordingly has been subject to intense

research. Despite numerous studies on model bacteria such as _Escherichia coli_ (_E. coli_), a debate persists on the intracellular organization and mobility of replisomes during DNA

replication. No active machinery has been identified to be responsible for the highly effective segregation of newly synthesized sister chromosomes, and the physical mechanisms at play have

remained incompletely understood. Replication in _E. coli_ initiates at a single genomic site called _oriC_, where two multi-protein complexes, called replisomes, are assembled. The

replisomes replicate the chromosomes bi-directionally away from _oriC_, and eventually, after duplicating the entire genome, meet again and terminate at specific Tus sites1,2 in the _ter_

region3. Two conflicting models have been proposed for the organization and positioning of the replisomes inside the cell (Fig. 1). The so-called ‘factory model’4 predicts that both

replisomes are co-localized together at a joint position near cell center, where the maternal DNA is replicated into daughter replicates of the two chromosome arms. Consequently, the newly

duplicated DNA is being pushed out toward the cell poles, resulting in proper chromosome segregation. By contrast, the “train track model”5 predicts that the two replisomes move

independently along the chromosome arms as they carry out their function. In the latter case, the replisomes are not necessarily positioned jointly at midcell, but they could of course very

well still be observed there, especially near the start of the replication process where the two replisomes both start from _oriC_. Since the two replisomes would move independently, the

train track model would require a separate mechanism to spatially drive chromosome segregation. Various groups have used fluorescent microscopy techniques to study DNA replication and

segregation in bacterial cells. Reyes-Lamothe et al.6 observed replisome activity at different positions in the opposing cell halves throughout different stages of the _E. coli_ cell cycle,

thus arguing against the factory model with joint replisomes at a stationary site. However, in a more recent study, very different results were reported by Mangiameli et al.7, again based on

fluorescently labelled replisomes in rod-shaped _E. coli_. Here, functional replisomes were observed to form a single focus proximal to the cell center throughout a major part of the cell

cycle, suggesting the formation of a replisome factory. Another major difference in literature possibly accounting for the conflicting replication models is the use of various _E. coli_

strains and various growth conditions. Cass et al.8 observed fluorescently labeled chromosomal loci to move toward midcell before segregation in MG1655 _E. coli_ cells with doubling times

<1 h. As segregation initiated, the dynamics of loci were universal and independent of genetic position, consistent with a factory model. It is known that the finer details of chromosome

morphology is different between the MG1655 and AB1157 strains9, while the short replication time also has an influence on the chromosome organization10,11. While the debate on the mobility

of the replisomes proceeds, it is furthermore unclear whether and how the replisomes are involved in chromosome segregation, which occurs simultaneously with the replication. Various models

were proposed to explain the segregation process. _E. coli_ bacteria show an initial rapid separation of the newly synthesized origins of replication12, similar to what happens in _Vibrio

cholerae_13 where the segregation is driven by the _ParABS_ segregation system. Yet, no such active protein system has been found in _E. coli_. It has been debated whether cell wall

confinement may play a role in the process of segregation. Early experiments14 in spherical _E. coli_ cells (lacking MreB filaments that maintain the rod-shape of the cell) indicated that

the rod-shape was not essential for proper chromosome segregation. Based on polymer simulations, Jun and Mulder15, however, proposed an entropy-driven segregation model, in which internal

repulsive entropic forces act on chromosomes to spontaneously demix them in rod shape cells, as the demixed polymer state is thermodynamically favored over the mixed one. Youngren et al.10

strengthened the idea that entropy demixing may play a role with experiments on fast-growing _E. coli_ that showed that, throughout segregation, chromosomes spatially self-organize as

branched-ring-polymers under rod-shape confinement, where minimization of the chromosome free energy can gradually drive newly synthesized sister chromosomes away from each other to opposing

cell halves and consequently push non-replicated DNA toward midcell. In this picture, replisome positioning near midcell may result from the segregation process, rather than be its cause.

Other groups have argued that mechanical strain rather than entropic forces are driving the chromosomal segregation16,17. Important questions on DNA replication thus remain unresolved, most

importantly whether replisomes are separate and mobile or joint and static relative to the nucleoid, and what drives chromosomal segregation. There are various reasons why the answers to

these questions varied in the studies reported so far. The aforementioned replisome visualization approaches differed in the types of fluorophores used, as well as in which components of the

replisomes were tagged. In population-based assays, additional uncertainties arose when determining the protein positions due to their variability at various stages of cell cycle. For

example, it is challenging to distinguish foci from newly initiated rounds of replication from single foci from ongoing rounds. Yet another technical challenge is the resolvability of

independent replisomes due to optical resolution limits and the very compact state of the nucleoid in rod-shaped _E. coli_. Here, we overcome these technical limitations by visualizing the

process of replication of an individual chromosome in _E. coli_ cells with an increased length or width. We achieve this by modifying the shape of cells using low concentrations of cell-wall

attacking antibiotics, and by using a temperature-sensitive dnaC allele to generate cells with initially only one chromosome. In these temperature-sensitive (dnaCts) cells, DnaC protein is

inactive above 40 °C, and, since DnaC protein is vital for loading the helicase (DnaB) onto the origin of replication18, cells cannot initiate a new round of replication, while they will

finish already initiated rounds. This enables us to synchronously initiate a single round of replication and eliminate the uncertainty whether observed foci in a single cell are from

temporally overlapping replication rounds. Cells can maintain a single chromosome, elongate, and reach longer sizes compared to normal rod-shaped cells (Supplementary Fig. 1), with

accordingly larger nucleoid sizes19, overcoming the diffraction limit to resolve separate foci. We use well-established monomeric fluorophores that avoid the problem that replisome

co-localization may result due to attracting forces between fluorescent tags. By initiating a single round of replication in the cells and tracking the number of replisome foci, we can

determine whether the replisomes move independently or remain co-localized. This allows us to directly visualize the separation of two single replisome foci after replication initiation. We

find that replisomes move away from each other, clearly moving along the nucleoid arms as they replicate the DNA. Furthermore, we, interestingly, observe that the cell’s capacity to

successfully segregate the replicated chromosomes towards daughter cells strongly depends on the cell width, indicating that entropic repulsion plays a role in DNA segregation. RESULTS

REPLISOMES LOAD NEAR THE MIDCELL AND MOVE AWAY FROM EACH OTHER To trace the position of the replisomes, we monitor a fluorophore-labelled key component of it, viz., the β-clamp (DnaN), which

serves as a marker for the location of the replication fork20. The factory and train-track models predict clearly different results for the number and position of the replisome foci

relative to the nucleoid (Fig. 1). According to the factory model, we would expect only a single replisome focus near midcell that is stationary throughout replication (Fig. 1c, d). By

contrast, the train-track model predicts an initially formed single focus that separates into two foci with a twice lower fluorescent intensity, that gradually move away from each other and

away from the midcell (Fig. 1a, b). We performed experiments with temperature-sensitive (dnaCts) _E.coli_ cells where both the β-clamps and the chromosome were fluorescently labelled (see

MM). At permissive temperatures (40 °C), dnaCts cells maintain a single chromosome while growing longer in contour length (Supplementary Fig. 1, Supplementary movie 1). By placing these

cells for a short time period (~10 min) at room temperature and then back to 40 °C, we could synchronously re-initiate replication in ~85% of all cells (Fig. 2, Supplementary Fig. 2). As

seen in Fig. 2a–c, cells placed at 40 °C maintained a single chromosome positioned at the center of the cell, as previously observed19. The intensity of the nucleoids was not homogeneous but

showed a remarkable heterogeneity with a pronounced periodic-like structure, as reported by Fisher et al.17. High-DNA intensity regions were observed to occur semi-periodically along the

nucleoid (Fig. 2d), where the number of the maxima was proportional to the length of the nucleoid. From the autocorrelation between the peaks, we estimate a periodicity of the undulation of

0.73 ± 0.15 μm (error bar denotes standard deviation (s.d.); _N_ = 30, Fig. 2e). This observed periodicity of the nucleoids is nontrivial, especially given that the period appears to be

independent of nucleoid length. Polymer modelling of chromosomes may elucidate this and it may be of interest to study whether this periodicity systematically varies as a function of cell

length or cell cycle, opening up new possibilities for future experiments. Note that the average cell length as well as the nucleoid length at the point of initiation of replication are

significantly expanded in these elongated cells at 40 °C (Fig. 2f, g). The average cell length and nucleoid length at 40 °C were 7.7 ± 0.2 and 3.3 ± 0.1 µm, respectively, (errors are

s.e.m.), and 4.7 ± 0.05 µm and 2.6 ± 0.05 µm (± s.e.m.), respectively, for cells grown at 30 °C. At both temperatures, we found a linear dependence between the nucleoid length and the cell

contour length, similar to other studies19,21 (Fig. 2h, which was universal and not dependent on the strain type of _E.coli_ used, nor of the labelling of the replisomes, Supplementary Fig. 

3). Note that earlier work by our group showed that the size of individual nucleoids in elongated non-replicating cells reaches a saturation point for cells larger than ~15 µm,19 but here we

do not reach this saturation regime since the cells in our experiments were replicating and were shorter in length. After this initial characterization of the nucleoids, we monitored the

positioning of replisome foci relative to the nucleoid and cell contour. After initiating a single replication round, a single replisome focus was observed to form in the middle of the

chromosomes, with a deviation from the cell center of, on average, 0.45 µm, which is ~5% of the average cell length (Fig. 2i, j). Shortly thereafter, i.e., in less than 15 minutes of

initiation (which is 12% of the cell cycle time of 124 minutes in these conditions), the focus was seen to split into two foci in more than 70% of all cells (_N_ = 147), each with an almost

twice lower fluorescent intensity (2.3 ± 0.2 lower intensity, ±s.d., _N_ = 20) (Fig. 2i, j, Supplementary movie 2). The splitting of the replisome foci was observed to occur independent of

the number of chromosomes (Supplementary Fig. 4) and independent of temperature (as it was also observed in cells grown at 37 °C, Supplementary Fig. 5). After >30 minutes of replication

initiation, the number of cells with two foci gradually started to decrease (Supplementary Fig. 6), indicating that the fluorophores gradually bleached (Supplementary Fig. 7) and/or the

replisomes disassembled after finishing the replication. These observations directly conform to the train track model, where the replisomes first assembled at oriC, showing only one focus at

replication initiation since their inter-distance was not resolved due to the diffraction limit, whereupon two foci became visible as the replisomes spatially separated, each replicating

the material of an opposing chromosome arm in a different half of the cell length. After completing their function, the replisomes moved back towards each other (which was observed in ~25%

of all replicating cells (_N_ = 84), whereas the rest bleached during the measurement or had no or multiple replication bubbles) and after finishing the replication (Fig. 2k) disassembled,

reducing the observed number of foci per cell at replication termination. INDEPENDENTLY MOVING REPLISOMES FORM A REPLICATION BUBBLE To visualize the replication bubble, we used the same

dnaCts cells with the labelled DNA and replisome, but now added the MreB-inhibiting drug A22. Since MreB is essential for maintaining the rod shape of _E.coli_ cells22, the addition of A22

(which inhibits the MreB polymerisation) results in cells that are significantly wider than normal rod-shaped cells14,23,25. Our earlier work showed that cells treated with A22 stay

physiologically healthy and active, while the single nucleoid adopts a toroidal configuration within the larger available cell volume25. Importantly, these cells allow to follow the dynamics

of the replisomes and the nucleoid upon re-initiating replication. To verify whether the replisomes indeed move along the chromosomal arms, we thus expanded the cells with the A22 drug and

visualized the replication as it started within the toroidal-shaped nucleoid. In about 80% of all replicating cells (_N_ = 167), we could once again clearly observe a single replisome focus

near the center of the cell, that quickly split into two lower intensity foci that had a 1.9 ± 0.2 lower intensity (±s.d. _N_ = 30, Supplementary Fig. 8). The foci moved away from each other

along the nucleoid arms while replicating the DNA. Interestingly, we observed that the position of the replisomes was closely associated with regions of higher DNA intensity: As the

replisomes moved along the circular nucleoid, the high-intensity DNA regions also moved with them (Fig. 3b). From analyzing the correlation between the foci position and the clusters size

(see M&M), we found that the single replisome foci (after splitting) were typically near DNA domains that contained up to 20% of the whole chromosome signal. These clusters contained on

average twice more DNA than any random DNA cluster (Supplementary Fig. 9). These high-intensity regions represent spots where replisomes are actively duplicating new chromosome material and

our experiments allowed us to track the process of DNA replication in real-time. Interestingly, the data revealed an extended period where the newly replicated DNA stayed cohesed to the

mother DNA before genuinely separating into a replication bubble. In other words, we observed that the sister nucleoids did not mutually segregate into two separate strands immediately after

the initiation of DNA replication, but instead they stayed mutually cohesed along their length for some time (Fig. 3b, panel II and III). This in line with some earlier observations in

rod-shaped cells12,17,26. This period of mutual cohesion of the replicated DNA was typically ~15 min. It was followed by a quick segregation of sister chromosomes, leading to the

visualization of a clearly opened replication bubble as shown in Fig. 3b panel IV. Despite having an increased cell size and a single nucleoid, the cells were capable of fully replicating

the DNA and finishing the replication cycle, as seen in Fig. 3e, f. During replication, the nucleoids underwent large structural rearrangements, evolving from a toroid to a split toroid, to

a figure-8 shape, to finally two separated toroidal-shaped nucleoids in daughter cells. The widened cells thus could still replicate and segregate the DNA and fully divide the mother to

daughter cells, showing that the MreB filaments and the associated rod shape of cells were neither necessary for proper cell division nor for chromosome segregation14. Summing up, the

widening the _E. coli_ cells allowed to visualize the structural evolution of the nucleoid during replication at a level of detail that was inaccessible to earlier studies. We observed that,

rather than pulling the DNA through a joint factory, the replisomes function independently and move along the chromosome arms while replicating the DNA (Fig. 3f, replisomes moved back

towards each other in ~33% of all replicating cells; _N_ = 160). The replicated DNA stayed cohesed to the mother DNA for ~15 min, after which a replication bubble opened and the replication

proceeded until a figure 8 and finally two separate daughter chromosomes were formed. CHROMOSOME SEGREGATION DEPENDS ON THE LATERAL CELL WALL CONFINEMENT To better understand the nucleoid

rearrangements, in particular the segregation of chromosomes, in the replication process during the cell cycle, we imaged an _E.coli_ strain with a HU-mYpet labelled chromosome and ori1 and

ter3 FROS arrays25 with widefield and SIM microscopy (Fig. 4; Supplementary Movies 3 and 4; Supplementary Fig. 10). Consistent with the experiments described above, about half of all

replications (48% out of _N_ = 453) displayed replication bubbles and figure-8 shapes, again confirming the train-track model for replisomes (Fig. 4b–d). We found that the Left-Ori-Right

orientation of chromosomes that was reported earlier in rod-shaped _E. coli_ cells21,22,23,24 was preserved in these widened cell too. We observed the origin of replication of single

nucleoids to duplicate near the midcell location (Fig. 4e), but this positioning was not necessary for replication initiation, as cells with two chromosomes could also duplicate their

origins distal from the midcell position (Supplementary Fig. 11). In contrast to earlier studies10,27,28,29,30, we observed that shortly after splitting, the Ori foci moved into a random

direction (Fig. 4f), towards a region within the cell cytosol that was not yet occupied by the nucleoid. This is a nontrivial finding, as previous studies suggested a fine-tuned mechanism

that would drive the replicated ori’s to move towards the cell poles30. In very wide cells, the two replicated Ori’s could initially even orient along the short axis of the cell whereas only

after some time, as the amount of replicated DNA locally increased, they would re-orient towards the long axis and the cell poles (Supplementary Fig. 12). This observation suggests a

possible role for the spatial confinement of the cell wall in orienting the newly replicated nucleoids towards the cell poles. When monitoring large cells during several hours of

replication, we observed that they encountered difficulties in segregating their chromosomes (Fig. 4g and Supplementary Fig. 13). Quantitatively, a threefold increase in the cell area (from

1.8 µm2 to 5.2 µm2, _N_ = 371) at the moment of replication initiation decreased the probability of successful replication more than twofold (Fig. 4g). Interestingly, increasing the number

of chromosomes in larger cells (by keeping the cells longer at room temperature), compensated for the loss of cell wall confinement and chromosomes did segregate properly (Supplementary Fig.

 14), similar to observations in earlier studies14,23. In rod-shaped cells grown at 30 °C, unsurprisingly, almost all cells (_N_ = 139) successfully segregated their chromosomes (mean cell

area 2.0 ± 0.6 µm2; error is s.e.m.). These data also indicate that the confinement exerted by the cell wall directs the sister chromosome segregation in _E. coli_ cells. Shortly after the

Ori foci splitting (typically around 15–20 min after replication initiation), the Ter foci moved towards the midcell location and stably positioned there for long periods of time (Fig. 4h,

Supplementary movie 4). That the Ter foci position typically coincided with the midcell location suggests a spatiotemporal correlation between Ter and the divisome components31. Once the Ter

focus duplicated, the foci quickly moved towards the respective cell halves enabling the septum to finish division of the cells and form two daughter cells (Fig. 4h). After division, both

chromosomes recovered the toroidal topology, consistent with the notion that it is the physiologically active form of the _E. coli_ chromosome25. As expected, inhibiting the supercoiling

homeostasis32 by perturbed the activity of TopoIV33 and gyrases34 upon administering Novobiocin drug reduced the capability of replicated chromosomes to timely separate35 with up to 30% of

cells (_N_ = 340) ending up with cohesed chromosomes that were seen to get stuck at the division septum (Supplementary Figs. 15 and 16). Our data thus show that the cell wall confinement

acting on the chromosomes is important for the segregation of daughter chromosomes and that there is a fine-tuned temporal correlation between the DNA replication and cell division.

DISCUSSION The process of DNA replication and its consecutive transfer from the mother cell to its progeny is one of the most intriguing processes in all living organisms. Surprisingly, even

for the best studied organism, such as the _E.coli_ bacterium, there is currently no unified view on the spatiotemporal control of DNA replication and segregation. Here, we addressed this

by monitoring the replication and segregating of single chromosome in shape-modified cells. The use of a temperature-sensitive strain was advantageous for two reasons: first, it enabled us

to synchronously initiate a single round of replication across the population of cells, and second, the nucleoids in these cells were elongated and thus permitted a higher spatial resolution

(Fig. 2). Most importantly, we observed separate replisomes that independently moved from the origin to the terminus of replication along each of the two arms of the circular chromosome

(Fig. 2k). We could visualize and study “text-book examples” of replication bubbles that provide direct evidence for the train track model describing the replication. Some early studies on

rod-shaped cells reported a single replisome focus near the midcell position pointing instead toward the factory model of DNA replication. We believe that the authors of these studies

observed a single replisome focus because the nucleoids were highly confined which prevented the replisomes from moving further apart than the optical diffraction limit (~200 nm). Indeed,

while in more recent work Mangiameli et al.7 observed a single replication focus in ~80% of all cells, they, interestingly, also observed ~20% of foci to split into two foci for short

periods of time, hinting that the replisomes could possibly spatially separate. Our experiments extend these studies and provide increased resolution to resolve that the factory model can be

rigorously ruled out. A second major finding from our results is that chromosome segregation in slowly growing _E. coli_ cells is found to be directed by the cell width. In rod-shaped

cells, we saw that the cells did segregate the chromosomes properly for a variety of cell sizes. Upon using the A22 drug to widen the cells with single chromosomes, however, the segregation

process partially impaired. After replication initiation at OriC, newly synthesized DNA mass started to build up until the emerging two daughter chromosomes started to detach from each

other, to, from there on, quickly segregate to form a replication bubble. The movement of the newly replicated DNA appeared to be driven by redistribution of DNA mass from strongly confined

towards less confined space, which explains the initially random movement of the Ori’s towards the available free space in the widened cells (Fig. 4f). When running into the confining cell

boundary along the short axis, the segregating DNA material was re-distributed toward unoccupied space which was mainly available towards the cell poles (Supplementary Fig. 12). This also

explains the observation that larger cells with single chromosomes had trouble segregating their chromosomes to the daughter cells (Fig. 4g, Supplementary Fig. 13). Interestingly, similar

results were observed by the lab of Jeff Errington and colleagues in cell wall deficient (l-form) _Bacillus subtilis_ cells, where forcing cells into narrow microfluidics channels increased

the efficiency of successful chromosome segregation36. Our experimental data are supportive of the theoretical model by Jun and Mulder that predicted that entropy can be the driver for

sister chromosome segregation15 given a particular (rod-shape) confinement for given DNA concentrations37. Indeed, we observed that increasing the DNA amount inside a given confinement (by

initiating multiple replication rounds in widened cells; Supplementary Fig. 14), one would recover a proper segregation of the chromosomes to the daughter cells. Taken together, our results

show that DNA replication in _E.coli_ proceeds as predicted by the train track model of independently moving replisomes and that cell wall confinement plays an important role in segregating

the nucleoids. METHODS STRAIN CONSTRUCTION All strains are derivatives of _E. coli_ K12 AB1157 strain and were constructed by P1 transduction38. To construct strain AJ2818 (_dnaN- mCherry,

hupA- mYPet:: frt, dnaC2 (ts):: aph frt_), mCherry–dnaN strain39, a kind gift from Nynke Dekker, was transduced with P1 phage derived from strain FW155140 (_hupA-mYPet::aph frt)_ for

endogenous HU labelling. The resulting strain was cured of antibiotic resistance by flippase expressed from pCP2041. Then the strain was transduced with P1 phage FW195718 (_dnaC2(ts)

ΔmdoB::aph:: frt_) to result in a DnaC temperature sensitivity. The final strain was selected for kanamycin resistance and temperature sensitivity and was subsequently cured of antibiotic

resistance by flippase. For experiments with Ori1/Ter3 labels, strain FW2179 (_ori1:: lacOx240::hygR, ter3::tetOx240::accC1 ΔgalK::tetR-mCerulean:: frt, ΔleuB::lacI-mCherry:: frt, hupA-

mYPet:: frt, dnaC2 (ts):: aph frt_)25, was used. To generate MG1655 dnaCts and AB1157 dnaCts strains described in Supplementary Fig. 3, we first transduced the wildtype strains with

_hupA-mYPet::aph_ from FW1551, then cured of kanamycin resistance using pCP20, and further transduced with _dnaC2 ΔmdoB::aph frt_ from FW195725. GROWTH CONDITIONS For experiments with linear

cells, we grew cells in liquid M9 minimum medium (Fluka Analytical) supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glycerol (Sigma-Aldrich), and 0.1% protein hydrolysate amicase (PHA)

(Fluka Analytical) overnight at 30 °C to reach late exponential phase. On the day of the experiment, the overnight culture was refreshed (1:100 vol) for 2 h on fresh M9 medium at 30 °C. We

then pipetted 1 μl culture onto a cover glass and immediately covered the cells with a flat agarose pad, containing the above composition of M9 medium as well as 3% agarose. The cover glass

was then placed onto a baseplate and sealed with parafilm to prevent evaporation. The baseplate was placed onto the microscope inside a 40 °C incubator for 2 h to stop the cells from

replicating and to let them grow longer. To reinitiate DNA replication, the baseplate was moved to room-temperature for 10 min before placing it back onto the microscope inside the 40 °C

chamber for imaging. To obtain circular chromosomes, we used the same protocol as described above, with minor changes: We grew cells in liquid M9 minimum medium (Fluka Analytical)

supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glycerol (Sigma-Aldrich), and 0.01% PHA (Fluka Analytical) overnight at 30 °C to reach late exponential phase. On the day of the experiment,

the overnight culture was refreshed (1:100 vol) for 2 h on fresh M9 minimal medium at 30 °C. We then pipetted 1 μl culture onto a cover glass and immediately covered the cells with a flat

agarose pad, containing the above composition of M9 medium, A22 (final 3 μg/ml), as well as 3% agarose. The cover glass was then placed onto a baseplate and sealed with parafilm to prevent

evaporation. The baseplate was placed onto the microscope inside a 40 °C incubator for 2.5 h to stop the cells from replicating and to let them grow into round shapes. To reinitiate DNA

replication, the baseplate was moved to room-temperature for 10 min before placing it back onto the microscope (inside 40 °C chamber) for imaging. For treatment of replicating cells with

Novobiocin, we first grew the cells in the presence of A22 as described above for 2.5 h to ensure they reach desired size and shape. Then we moved the baseplate to room-temperature for 10 

min and afterwards added 10 µl of Novobiocin (~50 µg/ml final) to the agarose pad during replication initiation phase. Finally the cells were moved back to 40 °C chamber and imaged.

STATISTICS AND REPRODUCIBILITY All samples were repeated at least in biological duplicates. Bacterial growth experiments in bulk. _E.coli_ cells were grown on a clear-bottom 96-well plate

(Nunc) with a final volume of 150 μl of the solution in each well. The plates were loaded into an Infinite 200Pro fluorescence plate reader (Tecan, Männedorf, Switzerland) and incubated at

30 °C in the presence of various concentrations of Novobiocin drug (25, 50, and 100 µg/ml). Samples were shaken with orbital agitation (2.5 mm amplitude) for a period of ~12 h. The cell

density was measured at 600 nm with 15 min intervals, measured in biological triplicates. FLUORESCENCE IMAGING Wide-field Z scans were carried out using a Nikon Ti-E microscope with a 100X

CFI Plan Apo Lambda Oil objective with an NA of 1.45. The microscope was enclosed by a custom-made chamber that was pre-heated overnight and kept at 40 °C. mCerulean was excited by SpectraX

LED (Lumencor) _λ_ex = 430-450 through a CFP filter cube (_λ_ex/_λ_bs/_λ_em = 426–446/455/460–500 nm). mYPet signal was excited by SpectraX LED _λ_ex = 510/25 nm through a triple bandpass

filter _λ_em = 465/25–545/30–630/60 nm. mCherry signals was excited by SpectraX LED _λ_ex = 575/25 through the same triple bandpass filter. Fluorescent signals were captured by Andor Zyla

USB3.0 CMOS Camera. For each channel, between 3 and 11 slices were taken with a vertical step size of 227 nm (in total up to 2.3 μm). Structured Illumination Microscopy imaging was carried

out using a Nikon Ti-E microscope and a SIM module. A 100X CFI Apo Oil objective with an NA of 1.49 was used. Samples were illuminated with 515 nm laser line and a Nikon YFP SIM filter cube.

mYPet, mCerulean, and mCherry signals of the same sample were also captured through wide-field imaging using a Nikon-Intensilight lamp. Filter cubes used for the wide-field imaging

corresponding to the SIM images were CFP filters (_λ_ex/_λ_bs/_λ_em = 426–446/455/460–500 nm), YFP filters (_λ_ex/_λ_bs/_λ_em = 490–510/515/520–550 nm), and RFP filters (_λ_ex/_λ_bs/_λ_em = 

540-580 / 585 / 592–668). Up to 19 slices were taken with a vertical step size of 100 nm (in total 1.8 μm). SIM image reconstruction was done by using NIS-Elements (version 4.51) software.

During image reconstruction, special care was taken to use the recommended parameters to avoid reconstruction artefacts. Furthermore, care was taken to check for photo-bleaching during image

acquisition (which was negligible), to minimize drift during imaging, and to avoid artifactual signatures in the Fourier transforms of the reconstructed images42 (Supplementary Fig. 17).

DECONVOLUTION Image stacks of 3–19 slices of Z stack in wide-field imaging were deconvolved using the Huygens Professional deconvolution software (Scientific Volume Imaging, Hilversum, The

Netherlands), using an iterative Classic Maximum Likelihood Estimate (CMLE) algorithm with a point spread function (PSF) experimentally measured using 200 nm multicolor Tetrabeads

(Invitrogen). The PSF of the single-frame non-deconvolved widefield images had a FWHM of 350 nm horizontally and 800 nm vertically. Deconvolution, to a great extent, reduced the out-of-focus

noise in the images, which also led to an improvement in lateral resolution. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary

linked to this article. DATA AVAILABILITY The source data underlying Fig. 2e, h are provided as a Source data file. The raw microscopy data supporting the findings of this manuscript (Figs. 

2–4, and Supplementary Figs. 1, 2, 5, 6, 7, 10, 11, 13, 14, 16 and 17) are freely available for download from 4TU.ResearchData repository

[https://data.4tu.nl/repository/uuid:23de478d-8d08-4149-abd6-3398cbe14847]. All data is available from the authors upon reasonable request. Source data are provided with this paper.

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successful super-resolution structured illumination microscopy. _Sci. Rep_. 5, 15915 (2015). Download references ACKNOWLEDGEMENTS We thank Nynke Dekker and Sumit Deb Roy for the kind gift of

replisome-labelled _E.coli_ strain, and them as well as David Sherratt for discussions. The work was supported by ERC Advanced Grant SynDiv (no. 669598) to C.D., and by the Netherlands

Organisation for Scientific Research (NWO/OCW), as part of the NanoFront and BaSyC programs. A.J. acknowledges support by the Swiss National Science Foundation (Grants P2ELP2_168554 and

P300P2_177768). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Van der Maasweg 9, 2629 HZ,

Delft, The Netherlands Aleksandre Japaridze, Christos Gogou, Jacob W. J. Kerssemakers, Huyen My Nguyen & Cees Dekker Authors * Aleksandre Japaridze View author publications You can also

search for this author inPubMed Google Scholar * Christos Gogou View author publications You can also search for this author inPubMed Google Scholar * Jacob W. J. Kerssemakers View author

publications You can also search for this author inPubMed Google Scholar * Huyen My Nguyen View author publications You can also search for this author inPubMed Google Scholar * Cees Dekker

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.J. and C.D. conceived and designed the project. A.J. and H.M.N. constructed the bacterial

strains. A.J., C.G., and H.M.N. did the microscopy experiments. J.K. led image analyses. All authors wrote the paper. C.D. supervised the project. CORRESPONDING AUTHOR Correspondence to

Cees Dekker. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks the anonymous

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