Chromosome and Replisome Dynamics in E. coli: Loss of Sister Cohesion Triggers Global Chromosome Movement and Mediates Chromosome Segregation

Experimental Approach

Synchronous cell cultures were obtained using a baby cell column (Experimental Procedures; Bates et al., 2005). Exponentially growing cells are affixed to glass beads via their flagella. These beads are packed into a column through which fresh medium is flowing and flagellin synthesis is turned off. Thereafter, newly divided (“newborn”) cells that lack flagella flow off the column. Eluted cells collected over ~1% of a generation time comprise a uniform population of newborn cells which, upon further incubation in growth medium, proceed synchronously through at least two ensuing rounds of cell division. Baby cell cultures are in steady state, unperturbed by the synchronization process (Bates et al., 2005). To obtain sufficient cells for analysis of multiple events at each of a large number of time points after birth, multiple samples were collected in rapid succession from a single column and then incubated for appropriately varying amounts of time prior to analysis. Such populations exhibit highly reproducible cell cycles. In independent experiments, cell doubling times and the timing of assayed events within the cell cycle vary by <10 min (below; data not shown).

Timing of Landmark Events at Doubling Time (Td) = 125 min

DNA replication was analyzed by flow cytometry. The percentage of cells undergoing bulk DNA replication at a particular time point is given by the percentage of cells with DNA contents intermediate between 1n and 2n (Figure 2A). The level of “S phase” cells increases to a maximum at t = 40 min and then decreases (Figure 2Ca). Septation is detected as appearance of indentations visible by phase contrast microscopy (Figure 2B). The percentage of septated cells increases to a maximum at t = 110 min and then decreases (Figure 2Ca). Cell division, manifested by increased cell concentration (Experimental Procedures), occurs at ~t = 120 min. (Figure 2Ca). Total interdivision time, given by the time between the first and second division cycles after elution, is 123–125 min (data not shown). Thus, actual cell “birth” precedes emergence of cells off the column by ~3–5 min.

For a synchronous cell population, the progression of events can be described more precisely by an approach used previously for analysis of synchronous yeast meiosis (e.g., Hunter and Kleckner, 2001). (1) Any particular chromosomal or cellular stage has a particular duration or “life span,” which can be calculated as the area under the corresponding primary curve (in % cells × time) divided by the total percentage of cells that go through the stage. (2) Given such a life span, the area under a primary curve can be converted to a “cumulative curve” describing the percentage of cells that have entered the stage in question as a function of time. (3) The percentage of cells that has exited each stage is also given by a parallel curve displaced along the time axis by one life span. (4) “Time of occurrence” of an event is defined as the time at which 50% of cells have entered (or exited) the corresponding stage. In cell populations eluted from the baby-cell column, ~80% of cells are proceeding through S phase and other identified stages as a single synchronous wave while the remaining ~20% are a minority subpopulation of asynchronous cells (Bates et al., 2005). Since the rise and fall in primary data curves represent essentially only the synchronous cells, life spans are calculated by dividing the area under the primary curve by the percentage of such cells, 81% for the current experiment. Correspondingly, the resulting cumulative curves represent the behavior of this entire synchronous cell population and, as a result, rise to 100% (Bates et al., 2005).

A representative synchronous cell population growing under “standard conditions” behaved as follows (Figures 2Cb and 2Cc). Bulk DNA replication lasted 46 min and extended from t = 17 min to t = 63 min. These times are accurate to within 5 min (see the Supplemental Data available with this article online). The septation stage lasted 25 min and extended from t = 98 min through t = 123 min, when it is terminated by cell division. By independent measurements, division occurred at t = 121 min (above). This correspondence confirms the accuracy of cumulative curve analysis.

Sister Chromosome Relationships Evolve in Four Discrete Phases

Four chromosomal loci were probed simultaneously by fluorescence in situ hybridization (FISH): the replication origin (ori); the gln and lac loci, located respectively at ~133 and ~1084 kb clockwise from the origin; and a terminus region segment (ter) located directly opposite ori, near the dif locus involved in dimer resolution (Figure 3A). Each cell was visualized as a two-dimensional image in a single focal plane (e.g., Figure 3B). The number of FISH-staining foci for each locus present was scored in 50 cells at each time point. Detection was 75%–90% efficient at each locus as determined by the percentage of newborn cells exhibiting a signal (data not shown).

In newborn cells (t = 0), each of the four probed loci appears primarily as a single staining focus; two-focus cells then appear, first for ori (filled and open turquoise circles represent two independent experiments), then for gln and lac, essentially simultaneously (filled orange and green circles), and finally for ter (filled magenta circles) (Figure 3C). The frequency of two-focus cells then decreases coordinately at all loci at the time of (and due to) cell division (Figure 3C).

A locus may be represented as a single focus either because it has not yet been replicated or because it has been replicated but the two sister chromosomes remain tightly colocalized (e.g., Niki et al., 2000). When the times of appearance of two-focus cells as defined by FISH are compared with replication timing as defined by flow cytometry (Figure 2), three separation transitions emerge (Figures 3C–3E) which define four stages in the evolution of sister chromosome relationships (Figure 3E).

The Sister Separation Transition Involves Nucleoid Splitting and Wide Separation of Corresponding Sister Regions

Cells examined above by FISH were also examined for bulk chromosome status by staining with the fluorescent dye 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI). The nucleoid initially appears as a single cylindrical object (Figure 3B, top left) but, at later times, is bilobed or “split.” Nucleoid splitting is not an artifact of sample preparation: the same transition can be observed, with the same timing after birth, in living cells (data not shown).

Split-nucleoid cells appear at about the same time as cells containing two gln or two lac foci, as seen in both primary data and corresponding cumulative curves (Figures 3C and 3D). These data further support occurrence of a single underlying transition in which corresponding sister regions become widely separated into two different halves of the cell. Correspondingly, inspection of individual cells shows that, among cells containing two gln foci, two lac foci, and a bilobed nucleoid, all (>95%) exhibit one gln focus and one lac focus in each nucleoid lobe (Figure 4D; discussed further below). Importantly, this correlation holds even at the early time points, when such cells are first appearing, as expected if the three events are truly concomitant.

Sequential ori splitting, nucleoid splitting, and ter splitting were confirmed in an independent experiment (Figure 3F).

Method of Positional Analysis

We next defined, as a function of time after cell birth, the spatial positions of origin and terminus foci and nucleoid boundaries as well as the numbers and position(s) of immnostaining foci of a replisome component, DnaX. In a single experiment (Figure 3F), 200 cells at each of 13 time points were analyzed. For each cell, the end nearest to a ter focus was designated as “left.” Relative to this orientation, the middle of the cell was determined and defined as (0,0). The position of each focus of interest was then marked and assigned x and y coordinates (Figure 4A, middle). Absolute focus positions were then normalized relative to total cell length and width, giving a corresponding set of “relative” positions (Figure 4A, bottom). Normalization corrects for significant variation in absolute cell lengths among cells of the same age/stage (Bates et al., 2005), thus giving much sharper positional distributions. It also facilitates monitoring of the 1/4 and 3/4 positions, which have special significance as the division sites in the next cell cycle. 31,000 data points were defined in this way.

Origin, Terminus, and Nucleoid Dynamics

At each time point, a majority of cells belongs to a particular class as defined by the “splitting transitions” described above (yellow line, Figure 3F). The overall pattern of ori, ter, and nucleoid dynamics is described by their positional distributions in these majority classes (Figure 4C). Cumulative curve analysis, which utilizes the data from all cells, gives precise timing of identified changes (Figure 4B).

DnaX/Replisome Dynamics

Wright and colleagues have shown that, in living cells, during DNA replication, foci of a DnaX-GFP fusion protein correspond to sites of DNA replication forks (personal communication). In the present study, foci of this same fusion protein were visualized using anti-GFP antibodies (Experimental Procedures). Since DnaX dynamics are not specifically correlated with sister-separation transitions, focus positions in all cells are presented separately (Figure 4C, right). These data, plus cumulative curve analysis (Figure 4B), reveal three new features of DnaX dynamics.

First, a prominent DnaX focus appears at midcell immediately following cell division (Figure 4C, t = 0–10 min). This midcell localization can occur even in the absence of any chromosome, as seen in anucleate cells formed in a partition-defective mutant (Figure 5A). DnaX appears at midcell prior to localization of ori. Interestingly, in DnaX/ori cofoci, DnaX has an average position exactly at midcell while ori occurs on average ~0.3 μm away, almost always on the “right” side of the cell, i.e., the side from which it would have approached (Figure 5B). Regular separation of ori and DnaX is not an artifactual result of image misalignment (Experimental Procedures). Given that replication initiates immediately after localization of ori to midcell (above), initiation occurs within the DnaX/ori midcell ensemble.

Second, a single midcell DnaX focus remains through initiation of DNA replication and into S phase, implying that clockwise and counterclockwise replication fork ensembles are colocalized in an apparent macromolecular complex. After ~40% of “S phase,” cells exhibit two DnaX foci, which occur at highly variable, apparently independent positions throughout the nucleoid (Figure 4C, t = 40–70 min; Figure 5C). This transition implies splitting of clockwise and counterclockwise replisomes and their forks. It does not represent initiation of the next round of replication, which does not occur until after division (above), and does not represent precocious assembly of complexes in preparation for the next cycle, because two-focus cells are virtually absent during the last 40 min of the cell cycle. Replisome splitting occurs ~10 min after ori splitting (Figures 4B and 4C). The same sequence of events is seen by progressive appearance and disappearance of cells containing the appropriate numbers of ori and DnaX foci or ter and DnaX foci (Figures 5D and 5E).

Third, after completion of bulk DNA replication but prior to appearance of two ter foci, cells containing a single DnaX focus are transiently observed. Such cells are prominent at t = 70, 80 min (Figure 4C) and arise ~15 min after exit from DNA replication (Figure 4B). Correspondingly, cells containing one DnaX focus appear at late times in cells containing two ori foci (Figure 5D, 2,1 (B)) and in cells containing one ter focus (Figure 5E, 1,1 (B)), followed by cells containing one ter focus and no DnaX foci and, finally, two ter foci and no DnaX foci (Figure 5E, 1,0 (B) and 2,0). Late single foci still occur in dispersed positions. We infer that replisome ensembles tend to be lost sequentially from the chromosome. Since ter is flanked by sites that block progression of forks that progress beyond ter (Coskun-Ari and Hill, 1997), sequential encounters of the faster fork and then the slower fork with one such site could explain this pattern.

Comparison of Cellular and Chromosomal Events at Different Slow Growth Rates

To understand whether and how the observed events are linked to the time of cell birth, to the cell cycle as a whole, and/or to one another, we examined events of interest at two additional growth rates, one slower and one faster than the standard conditions analyzed above (Td = 90 and 300 min, versus Td = 125 min). We also examined origin, nucleoid, and terminus positions at Td = 90 min to assess temporal linkages amongst spatial changes prior to and during the nucleoid splitting transition.

I. Sister Colocalization and Separation with Accompanying Global Chromosome Movement

We show above that sister chromosomes remain together and then separate in a single coordinate transition involving most or all of the thus-far-replicated regions. This behavior does not include the origin or the terminus, which behave as spatially and functionally distinct domains with respect to sister disposition (below). We infer that sisters are specifically linked, that this linkage (i.e., “cohesion”) is lost at the time of the nucleoid splitting transition, and that sisters are then separated by some type of active mechanism. These observations confirm earlier suggestions of cohesion by Hiraga and colleagues (Sunako et al., 2001). Our results may also explain the failure of other studies to detect cohesion (e.g., Roos et al., 2001): those studies examined relatively fast growth rates, and we find that the duration of cohesion varies inversely with growth rate. The observed sister separation transition is especially remarkable in two respects. First, splitting appears to occur simultaneously throughout a very extensive region of the genome. Second, it is accompanied by a global change in chromosome disposition: the splitting sister chromosome regions become widely separated from one another; moreover, dramatic changes in position are also observed for regions not involved in the splitting process, e.g., the origin and terminus.

II. Loss of Sister Cohesion Mediates Sister Chromosome Segregation, Analogously to Eukaryotic Prometaphase

The nucleoid splitting transition places sister chromosomes in a 2-fold symmetric, ter-in and ori-out configuration, with loci in the splitting regions occupying positions that correspond to their positions along the chromosome. Thus, the events of this transition solve the fundamental problem of sister chromosome segregation (Figure 1). We infer that loss of sister chromosome cohesion triggers global chromosome movement which in turn mediates sister chromosome segregation

These findings suggest that chromosome segregation is mediated by a global internal change within the chromosomes. They specifically exclude the two prevalent models for bacterial sister segregation (Introduction). The replisome extrusion model implies that sisters are never linked and requires long-lived association and spatial constraining of clockwise and counterclockwise replisomes. We find that, in E. coli, sisters are initially linked and there is no long-lived association of the two replisomes; moreover, association can be lost either before or after (and thus independent of) sister splitting. The “origins as centromeres” model implies that localization of the origins to the 1/4 and 3/4 positions precedes and leads sister separation. We find that the opposite is true: sister segregation is mediated by the nucleoid splitting transition which can precede ori separation and which, in any event, is required for origin localization (to the 1/4 position).

Interestingly, sister separation during the E. coli nucleoid splitting transition is closely analogous to sister separation during prometaphase of the eukaryotic mitotic cell cycle. In both cases, sisters are initially colocalized along their lengths and then separate, in a single coordinate transition, into a side-by-side configuration where sisters are disposed in a 2-fold symmetric relationship (Introduction; Figure 7C). Thus, the E. coli process could represent a primordial sister segregation mechanism to which microtubule-based processes were later added. Interestingly, also, mitotic prometaphase is a period of pronounced chromosome expansion, in accord with such expansion as the force driving sister separation (Kleckner et al., 2004).

III. Three Other Phases of Sister Separation

Separation of sister origins and appearance of two ter foci are spatially and functionally independent of one another and of the major sister splitting transition. The timing of ter replication is not established, but we favor the view that appearance of two ter foci represents splitting of already-replicated ter’s. When sister foci appear, they are symmetrically disposed about the site of the prior single ter focus, as expected for splitting of tethered ter regions. Further, ter is likely replicated well before two ter foci appear: replisomes are lost prior to appearance of two ter foci, and such loss likely requires either progression of one fork past ter to a fork-blocking site or convergence of two forks, which probably does not usually occur exactly at ter. For ter and for ori, colocalization/separation could represent either cohesion/loss of cohesion or independent capture and release by a common underlying determinant.

Sister relationships in origin-distal regions remain to be determined, but we suggest that, after the nucleoid-splitting transition, newly synthesized sister regions move directly into the two already-established nucleoid domains (above).

IV. Midcell

The 1/4 and 3/4 positions are the sites of midcell in the subsequent cell cycle. Specific localization of ori to these positions confirms their early demarcation under linear cell cycle conditions, as known for fast growth conditions, and also suggests that ori might be specifically attached to some cellular determinant. How these sites are marked and the significance of ori localization to these positions remain important mysteries.

Appearance of a DnaX focus at midcell shortly after division is the earliest postdivision marking of midcell known thus far, preceding ori localization (above) and septum component FtsZ, which arises at midcell after replication is in progress (Td = 200 min; Lau et al., 2003). ori foci become located at a nearly fixed distance to a specific side of the midcell DnaX focus, suggestive of a direct relationship between the two determinants. Other replisome and chromosome components are likely also present in this ensemble. Prior localization of DnaX might recruit oriC and/or ensure spatial coordination between replication initiation and ensuing replication fork development. Localization of oriC to midcell might be required for replication initiation and/or for regular coupling of initiation to the cell division program. After replication initiates, the midcell ensemble is disassembled in an ordered sequence, in apparent correlation with progression of DNA replication. The mechanism and functional significance of disassembly also remain to be determined.

Later in the cell cycle, ter arrives immediately adjacent to midcell, as a result of the nucleoid-splitting transition. ter seems to be specifically captured at the time of this relocalization because (1) there is little movement thereafter and (2) late-disappearing DnaX foci, which should be approaching the ter region, appear to move specifically toward midcell, as if tracking along to an anchored ter. After two ter foci appear, the ter nearest to midcell moves across to a symmetrical position on the opposite side, as described previously (Lau et al., 2003; Li et al., 2003). Importantly, terminus-related events at midcell are spatially and functionally distinct from ori/DnaX-related events. DnaX occurs exactly at midcell, and ori usually occurs to the “right” and ter usually occurs to the “left”. Moreover, ter localization may precede or follow disassembly of the ori/ DnaX ensemble according to growth rate.

V. In E. coli, Clockwise and Counterclockwise Replisomes Are Colocalized Only Transiently

In Caulobacter, cells undergoing DNA replication during a linear cell cycle always contain a single focus of replisome components (Viollier et al., 2004); similarly, in B. subtilis, such components exhibit dynamic variation between a single focus and two nearby foci (Migocki et al., 2004). In contrast, in linearly cycling E. coli cells, single replisome foci are lost partway through “S phase.” Thereafter, the two replisomes are well separated and independently disposed throughout the nucleoid. This pattern reflects dynamic and independent movement of the two as shown by time-lapse studies (A. Wright, personal communication; D.B., unpublished data). The observed movements could represent motion of the replisome relative to a fixed nucleoid, motion of the nucleoid with little relative motion of the replisome, or a mixture of the two.

VI. The Division Transition

The current study reveals previously undescribed features of E. coli chromosomes as they traverse the cell division transition. Immediately prior to division, sister nucleoids closely juxtaposed to midcell and their origins and termini occur at specific positions relative to the cell boundaries, apparently due to capture by underlying cellular determinants (above). Concomitant with division, sister nucleoids translate away from mid-cell such that they occupy symmetrical positions within their two about-to-be daughter cells. During this transition, the origin and the terminus retain their same positions relative to the nucleoid but change positions relative to the cell boundaries. These features strongly suggest that, during this transition, (1) the nucleoid is effectively “frozen” and (2) origins and termini are released from whichever determinants previously linked them to fixed cellular positions. Moreover, after division, the origin and the terminus both lose their strong localization and occur at many positions within their respective halves of the cell, which suggests that the nucleoid has become “unfrozen” concomitant with cell division. The origin then becomes localized on the non-ter side of the midcell DnaX ensemble as if it eventually drifts to and is captured by the midcell ensemble.

VII. Coordination of DNA Replication and Cell Division

Bacterial Strains and Growth Conditions

All experiments were performed in NK9387, CM735 (Wold et al., 1994) carrying an isopropyl β-D-thiogalactopyranoside (IPTG)-inducible sticky flagellin tethering construct (Ptac::fliC^st lacI^Q) plus a dnaX allele encoding a C-terminal dnaX-GFP fusion construct (gift of A. Wright; Bates et al., 2005). The GFP fusion has no detectable effect on either doubling time or DNA replication (data not shown). Cells were grown at 30°C in AB minimal media with 0.2% alanine, 0.2% succinate, or 0.05% glucose as indicated, and supplemented with tryptophan, histidine, methionine, and thiamine at 20 μg/ml each.

Time-Course Analysis

Preparation and use of the baby cell column are described by (Bates et al., 2005). After column equilibration, a series of samples were collected in rapid succession and then incubated for different appropriate periods of time (text). Sample volumes were proportionally scaled with growth rate (AB succinate, 10 ml; AB alanine, 5 ml; AB Glucose, 2 ml), about 2–3 × 10⁸ cells per sample. After the desired incubation time, one half of each sample was processed for FACS analysis (Bates et al., 2005) while the other half was processed for FISH/immunocytology (below).

Five Data Sets

Results presented for standard conditions (Td = 125 min) represent two independent sets of synchronized cell samples. Figures 2, 3F, 4, and 5 present results obtained from a single sample set I; Figures 3A–3E and 4D present results obtained from an independent sample set II. Timing of landmark events in set I are applicable to set II due to high reproducibility of kinetics among independent experiments performed under any given set of conditions (D.B. and N.K., unpublished data). Two additional sets of synchronized cells were obtained at different growth rates; Td = 90 and 300 min (Figure 6).

DNA Replication

The percentage of cells undergoing DNA replication was obtained by subtracting the percentage of cells containing full genome complements (either one or two fully replicated chromosomes) from the total. Since the DNA fluorescence histogram peaks corresponding to one and two chromosomes are partially obscured by cells in early and late stages of replication, these percentages were approximated by quantifying the “outer” half of the corresponding peaks (the sides of the peaks that do not overlap cells in the process of replication), and multiplying by 2 (details in the Supplemental Data available with this article online). This calculation was performed five independent times for each histogram and the results averaged.

Cell Concentration

Cell concentration was determined from the same FACS analyses used for analysis of replication (above) by comparing the rate of cell flow for each sample to that for a standard of known concentration.

FISH, DAPI, and Immunofluorescence

For FISH analysis, cells were fixed in 2.5% paraformaldehyde and hybridized with multiple fluorescently labeled probes (below; Supplemental Data). In sample set I and in samples representing other growth rates, DAPI was added at 150 μg/ml. In sample set II, one of the colors detected by multicolor FISH analysis overlaps significantly with DAPI fluorescence (below); thus, slides were first scored for FISH foci and then, after removal of coverslips, washed twice in 1× SSC, stained with DAPI, and scored for nucleoid status. For certain per-cell studies, these samples were also rescored for still-visible FISH foci. Note: late in the cell cycle, split sister nucleoids are well separated from one another (ca. 0.4 μm); in contrast, newly split nucleoids have a dumbbell appearance, with a prominent central connection, and thus are occasionally missed when the plane of focus runs through the connected region. This results in artificial flattening of the cumulative curve for nucleoid splitting.

FISH probes corresponded to 6 kb regions. Corresponding DNA fragments were obtained from plasmids by restriction digestion and gel purification (probe coordinates in the Supplemental Data), purified on a spin column (Qiagen), and chemically labeled with Alexa fluor (Ulysis DNA labeling system, Molecular Probes). Probe/ fluor combinations were ori/Alexa 594 and ter/Alexa 680 for all experiments except for the four-color FISH experiment (Figure 3), which used ori/Alexa 488, gln/Alexa 594, lac/Alexa 680, and ter/ Pacific Blue. The emission spectrum of the latter signal overlaps that of DAPI but, in favorable cases, was bright enough to be detected by a narrow band-pass filter that allowed five-color visualization (Figure 3B). When analyzed in combination with FISH, DnaX-GFP complexes were detected using anti-GFP antiserum immediately prior to in situ hybridization (Supplemental Data).

Fluorescence and phase contrast images were obtained using a Zeiss Axioplan2 microscope equipped with a Princeton CCD camera and analyzed using Metamorph software (Universal Imaging). Multiple fluorescent and phase contrast images taken for each field of cells were aligned to multiwavelength-emitting beads (Molecular Probes), placed in low concentration onto each slide as an internal control. At each time point in all experiments, 200 cells were analyzed for each feature of interest; the only exceptions were Figures 3A–3E and 6, Td = 90 min and 300 min, where 50 cells were analyzed per time point.