Replication and segregation of an Escherichia coli chromosome with two replication origins - PubMed (original) (raw)
Replication and segregation of an Escherichia coli chromosome with two replication origins
Xindan Wang et al. Proc Natl Acad Sci U S A. 2011.
Abstract
Characterized bacteria, unlike eukaryotes and some archaea, initiate replication bidirectionally from a single replication origin contained within a circular or linear chromosome. We constructed Escherichia coli cells with two WT origins separated by 1 Mb in their 4.64-Mb chromosome. Productive bidirectional replication initiated synchronously at both spatially separate origins. Newly replicated DNA from both origins was segregated sequentially as replication progressed, with two temporally and spatially separate replication termination events. Replication initiation occurred at a cell volume identical to that of cells with a single WT origin, showing that initiation control is independent of cellular and chromosomal oriC concentration. Cells containing just the ectopic origin initiated bidirectional replication at the expected cell mass and at the normal cellular location of that region. In all strains, spatial separation of sister loci adjacent to active origins occurred shortly after their replication, independently of whether replication initiated at the normal origin, the ectopic origin, or both origins.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
(A) Genetic map of the WT E. coli chromosome, with the left replichore in blue and the right replichore in orange. A zoom of th_e_ origin region shows the minimal initiation site oriC (258 bp) and its genetic context alongside the 5.1-kb and 1.2-kb DNA region, the segments that were relocated and/or deleted. oriZ (at 344 kb on the E. coli genetic map) is the site where the ectopic origin was inserted. The red and blue stars respectively indicate the insertion position of ori1 (lacO, at 3,908 kb) and R2 (tetO, at 366 kb) operator arrays used for chromosome localization. Replication termination sites (black dumbbell shapes), rRNA operons (black arrows), DnaA-binding DARS1 and DARS2 regions (812 kb and 2,967 kb, respectively), and the DnaA-binding cluster datA (3,494 kb) are shown, as is the dif site at which XerCD-FtsK recombination occurs. (B) Chromosome structure and representative newborn cells, with fluorescently marked ori1 (red) and R2 (blue) loci, of all three strains analyzed in detail. The replication arms inferred from the position of origins and terA and terC sites are represented by light gray arrows. Dashed lines indicate where replication and transcription are head-on. Lower: Doubling times (min; τ) in rich and minimal media, cell cycle parameters from flow cytometry (
Fig. S1
), and microscopy characterization (Figs. 3 and 4 and
Fig. S2
). B-period (min) is the time from birth to initiation of DNA replication, as measured by appearance of the replisome marker Ypet-DnaN. C-period (min) is the time of DNA synthesis assessed by replisome appearance to disappearance and by flow cytometry (C- plus D-period). D-period (min), the time from termination of DNA synthesis to division, was assessed by flow cytometry (C- plus D-period) and arithmetically as τ equal to the sum of B-, C-, and D-periods under our growth conditions. Cell volumes (in μm3) were measured by flow cytometry and direct microscopic measurement. During time-lapse analysis, cell doubling time on the agarose pads increased by 13% (oriC and oriC-oriZ strains) and 14% (oriZ); with a proportional increase in B-, C-, and D-periods, compared with growth in liquid medium. The values given have been compensated for this and reflect liquid growth values. The viabilities, determined as colony forming units per A600 were 1.95 × 109 (oriC) and 1.85 × 109 (oriC-oriZ and oriZ).
Fig. 2.
Synchronous initiation at both origins in oriC-oriZ cells. (A) Snapshot analysis of replisome (Ypet-DnaN) colocalization with genetic loci ori1 and R2, 10 min after initiation in temperature-sensitive dnaC2 derivatives of oriC, oriC-oriZ, and oriZ strains. Orange arrowheads indicate colocalization of replisome (green) and ori1 (red), and blue arrowheads indicate colocalization of replisome (green) and R2 (blue). Cell contours (white) have been added by using the phase-contrast images. The histograms show the proportion of different cell types in the three strains. Cells (n = 800–1,000) were analyzed for each strain. (B) Time-lapse analysis of replisome appearance in steady-state cells. A representative time-lapse image is shown for oriC-oriZ cells, in which replisome, ori1, and R2 are monitored simultaneously. Green arrowheads indicate replisome foci at initiation. The histograms show the number of replisome foci per cell within the time frame of Ypet-DnaN appearance, as observed by time-lapse microscopy. ori1 (lacO) was visualized by using LacI-mCherry (red); R2 (tetO) was visualized by using TetR-CFP (blue).
Fig. 3.
(A) Indicative time-lapse images of replisome (Ypet-DnaN) dynamics for oriC, oriC-oriZ, and oriZ cells growing on minimal medium with glycerol. Times (min), as well as the number of Ypet-DnaN foci in the corresponding frame, are indicated. (B) Cell cycle features of oriC, oriC-oriZ, and oriZ cells. Generation times in liquid were used to define the average time from birth to division. The mean replication C-period, as judged by the appearance and disappearance of Ypet-DnaN foci in time-lapse microscopy period in oriC (23 cells), oriC-oriZ (27 cells), and oriZ (24 cells), is labeled with green boxes, indicated with the number of resolvable foci (
Fig. S2_B_
). The times of replication initiation (Ini) and termination (Term) are indicated, with uncertainties indicated underneath by horizontal bars. The timings of ori1 and R2 separation, observed in time-lapse experiments (
Fig. S2_A_
), are labeled with empty red arrowheads (S, separation) and placed on top of the timeline with their uncertainties. The timings of ori1 and R2 replication were calculated on the basis of their genetic position and a mean replication rate for a 56-min C-period and is shown in empty blue arrowheads (R, replication), underneath the timeline with the uncertainties. In oriC-oriZ cells, 77% of cells with three replisomes had spatially separated ori1 and/or R2 sister foci (Fig. 4_A_). Times are in min.
Fig. 4.
A switch in loci segregation pattern in oriC-oriZ cells after replication of the smaller 1-Mb segment between oriC and oriZ. (A) Patterns of ori1 and R2 segregation in snapshots of the 14% (n = 2,472) oriC-oriZ cells containing three or four replisomes, and therefore have the 1-Mb fragment between oriC and oriZ still replicating (Left). Only 6% of cells have <_ori1 R2 ori1 R2_> or <_R2 ori1 ori1 R_2> genetic locus patterns, whereas 71% have sister loci adjacent and 23% are noninformative because sisters of neither locus have spatially separated. Later in the cell cycle (Right), when chromosome replication is complete as judged by the disappearance of the replisome foci (13% of 1,894 cells), 98% of cells had the <_ori1 R2 ori1 R2_> (69%) or <_R2 ori1 ori1 R_2> (29%) pattern. Data were analyzed from three independent experiments; SD shown in brackets. The same trends were observed in time-lapse analysis (
Fig. S2_A_
). The thick arrows show the switch in patterns (black, increase; gray, decrease). (B) Comparison of segregation patterns of fast-growing WT oriC cells (born with two sister oriC copies) versus slow-growing oriC-oriZ cells. After initiation, sister origins in the fast-growing cells segregate to the same daughter cell after cell division, giving rise to the pattern. In oriC-oriZ cells, sister origins initially adopt the same initial pattern, but switch to after replication of the 1-Mb fragment between oriC and oriZ is complete. Sister origins are shown in the same color (green or red), replication forks are shown in green triangles, and gray dashed line indicates division sites.
Fig. P1.
Replication and segregation patterns of WT E. coli cells (oriC), cells with two replication origins (oriC-oriZ), and cells with only the ectopic origin (oriZ). The circular chromosome is illustrated with black circles or relaxed circles, with rod-shaped cells of the same replication stage placed underneath. Gray dumbbells indicate the two innermost termination sites. The chromosome locus close to oriC (ori1) is labeled in red and that close to oriZ (R2) is labeled in blue. A locus is illustrated as an open circle when it is close to a functional origin and as a solid circle when it is not. For clarity, replisomes of the same cell are shown separately in green in a cell underneath. Black dashed lines indicate the colocalization of the replisomes and chromosome loci at initiation.
References
- Nielsen HJ, Li Y, Youngren B, Hansen FG, Austin S. Progressive segregation of the Escherichia coli chromosome. Mol Microbiol. 2006;61:383–393. -PubMed
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